Written by: Abby Stahl
Written By: Kaitlyn Sadtler
Original Article: Moser et al. Journal of Experimental Medicine 2019
The Gist of It:
Our body has amazing capabilities to defend itself from danger. Our immune cells fight off countless potential pathogens every day, while at the same time preventing the destruction of our own cells. However, when things go awry, we can see different illnesses that can broadly be defined as “autoimmune diseases”. These diseases have a variety of causes, including genetic, viral, or idiopathic (a fancy word that doctors use instead of “we don’t know”). The more we know about how our body naturally prevents autoimmune diseases, the more we can learn about how things may be unbalanced in patients with autoimmune conditions. Recent work from the Children’s Hospital of Philadelphia has shed some light on how our antibody-producing cells (called B cells) regulate themselves. B cells, named for the bursa of Fabricus (an organ in the chicken where B cells were originally discovered – your fun immunology fact of the day!), are stimulated by antigens, which are small pieces of proteins that, like barcodes on food at the grocery store, help the B cell identify the piece of protein and, if it identifies the piece of protein as foreign (aka not belonging to your body), produce the proper antibodies to neutralize the threat. In autoimmune diseases, B cells can start to produce antibodies that attack our body’s own proteins instead. One protein inside our cells that is called “Itch” controls the activation of B cells. Mice that lacked the protein Itch had higher numbers of activated B cells, which had increased sugar metabolism and energy consumption. Understanding these mechanisms of how our body keeps itself in check will help scientists develop new treatments to curb autoimmune diseases in a targeted manner as opposed to simply using broad-spectrum steroids, which is the current standard practice but does not impact the underlying problems to actually solve the problem causing the disease.
The Nitty Gritty:
Previous research has shown that Itch (an E3 ubiquitin ligase) can regulate autoimmunity. Itch knockout (KO) mice show autoimmune symptoms including the presence of autoantibodies (anti-dsDNA IgG). Moser et al. showed that in Itch KO mice, there were larger proportions of germinal center B cells (GL7+CD38−) and class switched non-germinal center B cells (GL7−) in the spleen and higher proportions of class-switched IgM and IgG1+ plasma cells in the bone marrow (BM). In a mixed BM chimera (using CD45.1 WT and CD42.2 Itch KO BM) there was a higher proportion of CD45.2+ Itch KO B cells. Through proteomic analysis of WT and Itch KO B cells that were stimulated with CpG, the authors showed higher expression of proteins associated with mTORC1 activation, cell cycle & E2F transcription factors as well as G2/M checkpoint proteins, and proteins associated with inflammatory responses to IFNγ and TNF. In accordance with the upregulation of cell cycle-associated proteins, Itch KO B cells had increased cell division in vitro when stimulated with anti-IgM or CpG when compared with a WT control (assessed via staining with a cell tracker dye). As expected based on the mTORC1 activation signature, Itch KO mice also displayed heightened glycolysis, which was determined by an increase in the extracellular acidification rate after stimulation with oligomycin in a Seahorse assay. After immunizing mice with NP-OVA, there were greater numbers of B18 germinal center B cells and NP+ plasma cells. The antibodies produced by this response were of higher affinity in the KO mouse than in the WT and displayed more mutation in the CDR1 gene region. Furthermore, when stained with a cell tracker dye and transferred in vivo, Itch KO B cells proliferated more after immunization and expressed higher levels of P-S6 (phosphorylated-S6) after a 1-hour ex vivo culture compared with WT B cells, suggesting Itch limits both cell division and mTORC1 activation.
Moser E.K., et al. “The E3 ubiquitin ligase Itch restricts antigen-driven B cell responses.” J Exp Med (2019): pii: jem.20181953.
Written By: Rebecca Tweedell
Original Article: Shimamura et al. Frontiers in Microbiology 2019
The Gist of It:
In people with healthy, fully functional immune systems, we don’t often hear about fungal infections. However, in people who are immunocompromised, including infants, the elderly, patients undergoing chemotherapy, and many others, fungal infections are much more common. One of these opportunistic fungal pathogens is Candida glabrata. The incidence of C. glabrata infections increased four-fold between 1992–1993 and 2008–2011, but little is known about how it achieves its virulence. One thing that is known is that fungi can use a process called autophagy to help them survive when nutrients are lacking, and some believe that this process may also be important for virulence. The word “autophagy” comes from “auto”, meaning “self”, and “phagein”, meaning “eat”; so autophagy = self eat. Kind of like in that saying “I’m so hungry my stomach is eating itself!”, during autophagy, cells break down some of their internal contents to recycle their building blocks to make new things that they need. Researchers at Nagasaki University have been studying how autophagy might play a role in C. glabrata’s virulence. They compared normal C. glabrata to a mutated strain that could not perform autophagy and found that the mutated strain did not grow as quickly under nutrient-limiting conditions. Additionally, this mutant did not handle other stresses, such as treatment with hydrogen peroxide, as well as normal fungi that could use autophagy did. Most importantly, in mouse models of C. glabrata infection, they found that the fungi that could not use autophagy did not infect mice as well as normal fungi did. This research highlights the importance of autophagy in not only the pathogen’s survival during stress, but also its ability to be a successful pathogen. Based on this work, treatments that block autophagy may be successful in treating C. glabrata infections, reducing the threat of these pathogens to those at risk.
The Nitty Gritty:
A C. glabrata strain that could not perform autophagy was created by deleting Atg1, a gene that encodes a protein in the ATG protein complex required for the induction of autophagy. The Cgatg1Δ strain was compared to the wild type (WT) and to a reconstituted strain that had ATG1 expression restored (Cgatg1Δ + CgATG1). Using spot assays in which the same number of cells from each strain was spotted on agar plates, researchers found that the Cgatg1Δ strain had growth defects in the presence of H2O2 and under nitrogen starvation conditions, while Cgatg1Δ + CgATG1 growth was comparable to that of WT. When treated with H2O2, the Cgatg1Δ cells had 60-fold higher levels of reactive oxygen species compared with WT and the Cgatg1Δ + CgATG1 strain, which likely contributed to its growth defect. In an ex vivo experiment, fungi were co-incubated with murine peritoneal macrophages, and the growth of the fungi was monitored. While all strains could be phagocytosed by the macrophages, the WT and Cgatg1Δ + CgATG1 strain were able to continue to grow successfully despite the phagocytosis, while the Cgatg1Δ strain could not. Finally, BALB/c mice were infected with C. glabrata in two different models to monitor the effect of autophagy on virulence. In the disseminated candidiasis model (C. glabrata injected intravenously), the Cgatg1Δ strain had fewer colony forming units (CFU) per organ in the liver and spleen but comparable numbers in the kidney when compared with WT. In the intra-abdominal candidiasis model (C. glabrata injected intra-abdominally), the Cgatg1Δ strain had fewer CFU per organ in the liver, spleen, and pancreas. Taken together, these results show that autophagy is critical for C. glabrata survival under nutrient-deficient and stress conditions, and autophagy plays a role in fitness/viability during infection.
Original Research Article: Shimamura, S., et al. “Autophagy-inducing factor Atg1 is required for virulence in the pathogenic fungus Candida glabrata.” Front Microbiol 10 (2019): 27.
Written By: Kaitlyn Sadtler