CD8αα intestinal intraepithelial lymphocytes derived from two thymic precursors seed the intestine in early life

TCRαβ CD4 CD8αα intestinal intraepithelial lymphocytes (CD8αα IEL) descend from thymic precursors. To better define this IEL precursor (IELp) population, we analyzed their maturation, localization, and emigration. Using rigorous lineage exclusion criteria, we defined two precursors among DN TCRβ thymocytes: a nascent PD-1 population and a T-bet population that accumulates with age. Both gave rise to intestinal CD8αα IEL upon adoptive transfer. In adult mice, PD-1 cells contained more self-reactive clones, localized to the cortex, and were dominant in S1PR1-dependent thymic egress. Gut homing α4β7 was already expressed by these IELp at a thymic stage. To understand the kinetics of CD8αα IEL seeding the intestine, we performed "timestamp" experiments: We crossed Cd4 with Rosa26 (stop-floxed tdTomato) mice. In these mice, tamoxifen or its metabolite 4-OHT permanently labels every CD4 expressing cell. As TCRαβ T cells (including CD8αα IEL) go through a CD4 CD8 stage during thymic development, a single dose of tamoxifen or 4-OHT will label thymic IEL precursors permanently, so that they can be tracked when seeding the gut. Our results indicate that these cells enter the intestine during a narrow time window in early life and that this influx is almost completely shut down by the age of 3 weeks. These data provide an important foundation for understanding the biology of this abundant population of barrier surface T cells

Intestine-specific deletion of microsomal triglyceride transfer protein increases mortality in aged mice

BACKGROUND:Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8-10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. METHODS:Aged (20-24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. RESULTS:In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. CONCLUSIONS:Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice

Microfluidic Amperometric Sensor for Analysis of Nitric Oxide in Whole Blood

Standard photolithographic techniques and a nitric oxide (NO) selective xerogel polymer were utilized to fabricate an amperometric NO microfluidic sensor with low background noise and the ability to analyze NO levels in small sample volumes (~250 μL). The sensor exhibited excellent analytical performance in phosphate buffered saline, including a NO sensitivity of 1.4 pA nM−1, a limit of detection (LOD) of 840 pM, and selectivity over nitrite, ascorbic acid, acetaminophen, uric acid, hydrogen sulfide, ammonium, ammonia, and both protonated and deprotonated peroxynitrite (selectivity coefficients of −5.3, −4.2, −4.0, −5.0, −6.0, −5.8, −3.8, −1.5, and −4.0 respectively). To demonstrate the utility of the microfluidic NO sensor for biomedical analysis, the device was used to monitor changes in blood NO levels during the onset of sepsis in a murine pneumonia model

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database

Microbial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The ‘Biomes of Australian Soil Environments’ (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database

Background: Microbial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The 'Biomes of Australian Soil Environments' (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function. Findings: BASE currently provides amplicon sequences and associated contextual data for over 900 sites encompassing all Australian states and territories, a wide variety of bioregions, vegetation and land-use types. Amplicons target bacteria, archaea and general and fungal-specific eukaryotes. The growing database will soon include metagenomics data. Data are provided in both raw sequence (FASTQ) and analysed OTU table formats and are accessed via the project's data portal, which provides a user-friendly search tool to quickly identify samples of interest. Processed data can be visually interrogated and intersected with other Australian diversity and environmental data using tools developed by the 'Atlas of Living Australia'. Conclusions: Developed within an open data framework, the BASE project is the first Australian soil microbial diversity database. The database will grow and link to other global efforts to explore microbial, plant, animal, and marine biodiversity. Its design and open access nature ensures that BASE will evolve as a valuable tool for documenting an often overlooked component of biodiversity and the many microbe-driven processes that are essential to sustain soil function and ecosystem services

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus

TCRαβ+ CD8α+CD8β–intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely β2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+. However, selective depletion of β2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate

Phenotypic T Cell Exhaustion in a Murine Model of Bacterial Infection in the Setting of Pre-Existing Malignancy

<div><p>While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8⁺ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with <i>Listeria monocytogenes</i>, and antigen-specific CD8⁺ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8⁺ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8⁺ T cells and total CD4⁺ and CD8⁺ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8⁺ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8⁺ T cell functionality and differentiation.</p></div

Myosin Light Chain Kinase Knockout Improves Gut Barrier Function and Confers a Survival Advantage in Polymicrobial Sepsis

Abstract Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the perijunctional actin-myosin ring. Myosin light chain kinase (MLCK) phosphorylates the myosin regulatory light chain, resulting in increased permeability. The purpose of this study was to determine whether genetic deletion of MLCK would alter gut barrier function and survival from sepsis. MLCK−/− and wild-type (WT) mice were subjected to cecal ligation and puncture and assayed for both survival and mechanistic studies. Survival was significantly increased in MLCK−/− mice (95% versus 24%, p < 0.0001). Intestinal permeability increased in septic WT mice compared with unmanipulated mice. In contrast, permeability in septic MLCK−/− mice was similar to that seen in unmanipulated animals. Improved gut barrier function in MLCK−/− mice was associated with increases in the tight junction mediators ZO-1 and claudin 15 without alterations in claudin 1, 2, 3, 4, 5, 7, 8 and 13, occludin or JAM-A. Other components of intestinal integrity (apoptosis, proliferation and villus length) were unaffected by MLCK deletion, as were local peritoneal inflammation and distant lung injury. Systemic IL-10 was decreased greater than 10-fold in MLCK−/− mice; however, survival was similar between septic MLCK−/− mice given exogenous IL-10 or vehicle. These data demonstrate that deletion of MLCK improves survival following sepsis, associated with normalization of intestinal permeability and selected tight junction proteins