A Single-Cell Sequencing Guide for Immunologists

In recent years there has been a rapid increase in the use of single-cell sequencing (scRNA-seq) approaches in the field of immunology. With the wide range of technologies available, it is becoming harder for users to select the best scRNA-seq protocol/platform to address their biological questions of interest. Here, we compared the advantages and limitations of four commonly used scRNA-seq platforms in order to clarify their suitability for different experimental applications. We also address how the datasets generated by different scRNA-seq platforms can be integrated, and how to identify unknown populations of single cells using unbiased bioinformatics methods

mTORC2/Akt activation in adipocytes is required for adipose tissue inflammation in tuberculosis

BACKGROUND: Mycobacterium tuberculosis has co-evolved with the human host, adapting to exploit the immune system for persistence and transmission. While immunity to tuberculosis (TB) has been intensively studied in the lung and lymphoid system, little is known about the participation of adipose tissues and non-immune cells in the host-pathogen interaction during this systemic disease. METHODS: C57BL/6J mice were aerosol infected with M. tuberculosis Erdman and presence of the bacteria and the fitness of the white and brown adipose tissues, liver and skeletal muscle were studied compared to uninfected mice. FINDINGS: M. tuberculosis infection in mice stimulated immune cell infiltration in visceral, and brown adipose tissue. Despite the absence of detectable bacterial dissemination to fat tissues, adipocytes produced localized pro-inflammatory signals that disrupted adipocyte lipid metabolism, resulting in adipocyte hypertrophy. Paradoxically, this resulted in increased insulin sensitivity and systemic glucose tolerance. Adipose tissue inflammation and enhanced glucose tolerance also developed in obese mice after aerosol M. tuberculosis infection. We found that infection induced adipose tissue Akt signaling, while inhibition of the Akt activator mTORC2 in adipocytes reversed TB-associated adipose tissue inflammation and cell hypertrophy. INTERPRETATION: Our study reveals a systemic response to aerosol M. tuberculosis infection that regulates adipose tissue lipid homeostasis through mTORC2/Akt signaling in adipocytes. Adipose tissue inflammation in TB is not simply a passive infiltration with leukocytes but requires the mechanistic participation of adipocyte signals

Center on Disability Studies eNewsletter, June 2023

Welcome to our summer newsletter. In this issue we highlight many events and happenings sponsored by CDS during June and July that you don’t want to miss out on. Disability Pride Month is also celebrated each year in July. Disability Pride initially started as a day of celebration in 1990, the year that the Americans with Disabilities Act (ADA) was signed into law. It is also an opportunity to raise awareness about improving access and inclusion. The first official Disability Pride celebration occurred in 2015 to commemorate the ADA’s 25th anniversary and the Disability Pride Flag was originally designed in 2019 by Ann Magill, who with feedback within the disabled community, refined its visual elements in 2021 to be more accessible. You can read more about how the disability pride flag helps increase the community’s visibility at https://go.hawaii.edu/qEX

Inhibition of TIR Domain Signaling by TcpC: MyD88-Dependent and Independent Effects on Escherichia coli Virulence

Toll-like receptor signaling requires functional Toll/interleukin-1 (IL-1) receptor (TIR) domains to activate innate immunity. By producing TIR homologous proteins, microbes inhibit host response induction and improve their own survival. The TIR homologous protein TcpC was recently identified as a virulence factor in uropathogenic Escherichia coli (E. coli), suppressing innate immunity by binding to MyD88. This study examined how the host MyD88 genotype modifies the in vivo effects of TcpC and whether additional, TIR-domain containing proteins might be targeted by TcpC. In wild type mice (wt), TcpC enhanced bacterial virulence, increased acute mortality, bacterial persistence and tissue damage after infection with E. coli CFT073 (TcpC+), compared to a ΔTcpC deletion mutant. These effects were attenuated in Myd88−/− and Tlr4−/− mice. Transcriptomic analysis confirmed that TcpC inhibits MYD88 dependent gene expression in CFT073 infected human uroepithelial cells but in addition the inhibitory effect included targets in the TRIF and IL-6/IL-1 signaling pathways, where MYD88 dependent and independent signaling may converge. The effects of TcpC on bacterial persistence were attenuated in Trif −/− or Il-1β −/− mice and innate immune responses to ΔTcpC were increased, confirming that Trif and Il-1β dependent targets might be involved in vivo, in addition to Myd88. Furthermore, soluble TcpC inhibited Myd88 and Trif dependent TLR signaling in murine macrophages. Our results suggest that TcpC may promote UTI-associated pathology broadly, through inhibition of TIR domain signaling and downstream pathways. Dysregulation of the host response by microbial TcpC thus appears to impair the protective effects of innate immunity, while promoting inflammation and tissue damage

Macrophage polarisation associated with atherosclerosis differentially affects their capacity to handle lipid

Background and aims Lipid-rich foam cell macrophages drive atherosclerosis via several mechanisms, including inflammation, lipid uptake, lipid deposition and plaque vulnerability. The atheroma environment shapes macrophage function and phenotype; anti-inflammatory macrophages improve plaque stability while pro-inflammatory macrophages promote rupture. Current evidence suggests a variety of macrophage phenotypes occur in atherosclerotic plaques with local lipids, cytokines, oxidised phospholipids and pathogenic stimuli altering their phenotype. In this study, we addressed differential functioning of macrophage phenotypes via a systematic analysis of in vitro polarised, human monocyte-derived macrophage phenotypes, focussing on molecular events that regulate foam-cell formation. Methods We examined transcriptomes, protein levels and functionally determined lipid handling and foam cell formation capacity in macrophages polarised with IFNγ+LPS, IL–4, IL–10, oxPAPC and CXCL4. Results RNA sequencing of differentially polarised macrophages revealed distinct gene expression changes, with enrichment in atherosclerosis and lipid-associated pathways. Analysis of lipid processing activity showed IL–4 and IL–10 macrophages have higher lipid uptake and foam cell formation activities, while inflammatory and oxPAPC macrophages displayed lower foam cell formation. Inflammatory macrophages showed low lipid uptake, while higher lipid uptake in oxPAPC macrophages was matched by increased lipid efflux capacity. Conclusions Atherosclerosis-associated macrophage polarisation dramatically affects lipid handling capacity underpinned by major transcriptomic changes and altered protein levels in lipid-handling gene expression. This leads to phenotype-specific differences in LDL uptake, cellular cholesterol levels and cholesterol efflux, informing how the plaque environment influences atherosclerosis progression by influencing macrophage phenotypes

CXCR4 identifies transitional bone marrow premonocytes that replenish the mature monocyte pool for peripheral responses

It is well established that Ly6C(hi) monocytes develop from common monocyte progenitors (cMoPs) and reside in the bone marrow (BM) until they are mobilized into the circulation. In our study, we found that BM Ly6C(hi) monocytes are not a homogenous population, as current data would suggest. Using computational analysis approaches to interpret multidimensional datasets, we demonstrate that BM Ly6C(hi) monocytes consist of two distinct subpopulations (CXCR4(hi) and CXCR4(lo) subpopulations) in both mice and humans. Transcriptome studies and in vivo assays revealed functional differences between the two subpopulations. Notably, the CXCR4(hi) subset proliferates and is immobilized in the BM for the replenishment of functionally mature CXCR4(lo) monocytes. We propose that the CXCR4(hi) subset represents a transitional premonocyte population, and that this sequential step of maturation from cMoPs serves to maintain a stable pool of BM monocytes. Additionally, reduced CXCR4 expression on monocytes, upon their exit into the circulation, does not reflect its diminished role in monocyte biology. Specifically, CXCR4 regulates monocyte peripheral cellular activities by governing their circadian oscillations and pulmonary margination, which contributes toward lung injury and sepsis mortality. Together, our study demonstrates the multifaceted role of CXCR4 in defining BM monocyte heterogeneity and in regulating their function in peripheral tissues