How can fungus secretions help treat gout?

Written by: Ken Estrellas

Original Article: Wu et al. Archives of Biochemistry and Biophysics 2019
The Gist of It:
Gout, once known as “the disease of kings,” is an acute inflammatory disease that most people experience as sharp pains and swollen joints, commonly near the big toe. Poor diet, including excess consumption of alcohol, meat, and seafood, are associated with elevated levels of uric acid in the blood. As a result, uric acid crystals can form in joints and tendons, leading to the inflammation and pain of gout. Although many common treatments for gout are focused on lowering levels of uric acid in the bloodstream, some drugs such as over-the-counter painkillers are focused on reducing inflammation and symptoms in joints. One approach studied in a recent paper by Wu and colleagues evaluated the role that chaetocin, a compound secreted by the Chaetomium species of fungus, might play in treating gout. Chaetocin has been studied as an antibiotic and an anti-cancer drug, but in this study, the researchers demonstrated that it may also reduce symptoms of inflammation associated with gout. Macrophages, a type of immune cell that makes the symptoms of gout worse, were isolated from mouse bone marrow and used to test the effects of chaetocin. Experiments revealed that chaetocin reduced the amount of the cytokine interleukin-1 beta (IL-1β), which is associated with inflammation, released into the blood. Additionally, chaetocin appeared to reduce the levels of enzymes that facilitate glycolysis, a process that macrophages go through during gout. Finally, in mice with elevated levels of uric acid leading to gout-like symptoms, treatment with chaetocin appeared to reduce inflammation, foot swelling, and sensitivity to pain. Taken together, these results suggest that fungal-derived compounds such as chaetocin may help relieve the symptoms of gout by holding back the function of cells that promote inflammation, potentially making life easier for people who might suffer from “the disease of kings.” (Previously thought to only affect those who had access to rich foods – such as nobility — now we know otherwise!)
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Fungal secretions may prevent gout symptoms by preventing macrophages from spreading inflammatory signals.

The Nitty Gritty
In this study, Wu et al. demonstrated the potential utility of the fungal-derived (from Chaetomium spp.) thiodioxopiperazine antibiotic chaetocin in ameliorating symptoms of acute pain caused by elevated serum urea levels and inflammatory reactions to deposits of soluble monosodium urate (MSU) crystals in joints and tendons, a condition commonly known as gout. Murine bone marrow-derived macrophages (BMDMs) isolated from wild-type C57BL/6 mice were pre-treated with chaetocin and subsequently primed to release IL-1β with lipopolysaccharide and challenged with MSU. BMDMs pre-treated with chaetocin released lower concentrations of IL-1β. This reduction was linked to inhibitory effects on the NLRP3 inflammasome; chaetocin-treated cells showed lower levels of transcript and protein expression of the ASC adapter and multiple forms of caspase-1 (the immature proform and the cleaved form), both key components of the NLRP3 inflammasome, which mediates IL-1β secretion. Chaetocin was also demonstrated to inhibit the release of HIF-1α and hexokinase 2, key components of the glycolytic pathway associated with macrophage activation. Finally, mice that were pre-treated with 0.25 mg/kg chaetocin once daily for 3 days responded more favorably to challenge with 6 mg/mL MSU IP compared to non-treated mice; pre-treated mice showed significantly decreased expression of key NLRP3 inflammasome components, decreased hyperalgesia, and decreased serum IL-1β levels. Taken together, these results suggest a potential alternative strategy for the treatment of gout symptoms differing from traditional uric acid-lowering compounds such as allopurinol or over-the-counter treatments such as NSAIDS to reduce gout-induced pain.
Original Research Article: Wu, M., et al. “Chaetocin attenuates gout in mice through inhibiting HIF-1α and NLRP3 inflammasome-dependent IL-1β secretion in macrophages.Arch Biochem Biophys 670 (2019): 94–103.

Special delivery! How “deliveries” from Leishmania parasites influence the cells around them

Written By: Rebecca Tweedell

Original Article: Barbosa et al. Frontiers in Microbiology 2019
The Gist of It:
With the warm weather of summer fading away, many people are happy to see the insects going away, too. In addition to buzzing around our food and potentially stinging or biting us, several insects can carry disease. One such disease that affects people throughout the Americas, the Mediterranean basin, the Middle East, and Central Asia is cutaneous leishmaniasis, which causes raw skin sores that can lead to permanent scarring and deformity. Leishmania amazonesis is one of the species of Leishmania parasites responsible for this disease, and they are transmitted from person to person through the bite of the sandfly. While there are treatments for leishmaniasis, the L. amazonesis species often has a natural resistance to these drugs. Scientists at the Universidad Federal de Sao Paulo in Brazil have been working to understand more about L. amazonesis in an effort to find new ways to treat these infections. They recently focused in on the extracellular vesicles released from these parasites. Extracellular vesicles are cell-derived structures that can bleb off and be released into the surrounding environment. Cells can use these extracellular vesicles to communicate with surrounding cells and even to deliver cargo, such as proteins that would make the receiving cell resistant to drugs. The researchers found that L. amazonesis parasites were releasing extracellular vesicles into their environment, and that these extracellular vesicles could perform different functions depending on what cell type they encountered. When two different types of immune cells, bone marrow-derived macrophages and B-1 cells, were exposed to the extracellular vesicles, they reacted differently. Importantly, though, they both reacted in a way that would favor the parasite’s survival. The researchers went on to show that in a mouse model of leishmaniasis, mice that were given extracellular vesicles in addition to their infection had worse skin sores and higher numbers of parasites than mice that were infected without the addition of extracellular vesicles. Understanding more about how L. amazonesis infects and how it uses extracellular vesicles to improve its pathogenicity will help identify new ways to fight these parasites.

Extracellular vesicles from L. amazonesis parasites influence different immune cells to respond differently, ultimately allowing the parasites to multiply.

The Nitty Gritty:
The researchers began by determining whether extracellular vesicles (EVs) were released from L. amazonesis promastigotes after various amounts of time at 26°C (the average temperature inside the sandfly), 34°C, and 37°C. Nanoparticle tracking analysis showed that EVs were released over time at all 3 temperatures, but the number of particles released at 26°C was significantly higher. EVs released at all 3 temperatures were similar in size, averaging approximately 180 nm. Dot blot and ELISA analysis of the EVs revealed that they contained proteins with known immunomodulatory properties, GP63 and LPG. Bone marrow-derived macrophages (BMDMs) and B-1 cells were exposed to EVs obtained at the 3 different temperatures, and cytokine secretion was measured by ELISA. In BMDMs, TNFα secretion was not affected, IL-6 secretion was increased in response to EVs from parasites grown at 26°C and 37°C, and IL-10 secretion was only increased in response to EVs from parasites grown at 26°C. Interestingly, in B-1 cells, secretion of IL-6, IL-10, and TNFα were decreased in response to EVs from all 3 temperatures, showing that EVs can have different effects depending on the type of cell they encounter. To determine the significance of these in vitro findings in vivo, BALB/c mice were infected with L. amazonesis either with or without the addition of EVs from parasites grown at 26°C (mimicking the EVs that would be present during transmission from the sandfly). Co-injection of EVs with the parasites increased the number of parasites present 7 weeks post-infection and enhanced the progression of lesions. Overall, these findings show that EVs from L. amazonesis can play a key role in the parasite’s pathogenesis.
Original Research Article: Barbosa, F.M.C., et al. “Extracellular vesicles released by Leishmania (Leishmania) amazonesis promote disease progression and induce the production of different cytokines in macrophages and B-1 cells.” Front Microbiol 9 (2018): 3056.

To Jurkat or not to Jurkat: Differences in primary and immortal T cells

Written by: Kaitlyn Sadtler

Original Article: Colin-York et al. Journal of Cell Science 2019
The Gist of It:
There are many things that affect the way our immune system functions. One of those is the strength of the connection, or immunologic synapse, between two key immune cells, a T cell and an antigen presenting cell. Through this synapse, the T cell learns from the antigen presenting cell what pathogen it is facing by being presented (or shown) the antigen (which is a small piece of that pathogen). To improve our understanding of the immune system, it is important to use models that preserve this immunologic synapse. When studying our immune system, researchers can use a variety of models and tools, from looking at patient samples to considering animal models to using cells in a dish. When using cells in a dish, there are even more options, including cells freshly isolated from humans or animals, as well as so-called immortalized cells or cell lines. Immortalized cells were once normal cells living in a human or an animal but have been transformed so that they can live indefinitely outside the body in a tissue culture dish. One specific type of immortalized cell is the Jurkat. Jurkats were made using T cells collected from a 14 year old boy in the 1970’s. This boy had a disease called T-cell leukemia, which is a type of blood cancer. The leukemic T cells have a variety of mutations but still retain important T-cell functions. Recently, researchers looked at the formation of an immunologic synapse in primary T cells (cells directly collected from humans or animals) versus immortalized Jurkat cells. They focused specifically on a protein that is responsible for cell structure and movement known as actin. Actin can form long rods or filaments that help move the cell membrane (both on a large scale to move the cell, and on a smaller scale to make tiny protrusions like those used in immunologic synapses). Scientists discovered that there were differences between Jurkats and primary T cells. In Jurkats (which are bigger than their primary T cell counterparts) the actin looked different at the synapse; actin was more concentrated or tightly packed in primary cells and more diffuse in Jurkats. Additionally, the two cell types used different mechanisms for the structure and function of their actin filaments, with the Jurkats relying more heavily on actin turnover, in which individual pieces of actin hop on or hop off the filament on their own, and primary cells relying on a protein called myosin-II. Seeing these differences in primary versus immortalized cells shows us that we might need to re-think our assumptions about T cells and what conclusions we can draw from different models. Studies like these help scientists truly understand their data and what implications their experiments have on our understanding of human health and disease.
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Primary (taken directly from a human or animal) and Jurkat T cells (immortalized, derived years ago from cancer) have different cytoskeletal dynamics (the bones and muscles of a cell)

The Nitty Gritty:
Colin-York et al. analyzed the differences between (1) Jurkat T cells, (2) human CD4+ T cells, and (3) murine CD4+ T cells. Using total internal reflection (TIRF) and structured illumination microscopy (SIM) they noted distinct differences when cells were activated on a synthetic planar lipid bilayer system (dipalmitoylphosphatidylcholine [DOPC] + anti-CD3). Although calcium flux remained the same (determined by Fluo-4 AM labeling), actin dynamics were altered. In both human and mouse primary derived T cells, there was a distinct punctate actin staining that was absent in Jurkat T cells. Lamelipodial leading edge curvature and dynamics were greater in primary murine T cells when compared with Jurkat T cells, though this did not have a significant change upon the velocity of TCR (T cell receptor) microcluster movement. When modulating actin behavior with several compounds, researchers found that in primary T cells, the flow of F-actin was largely dependent upon myosin-II (determined by blocking with blebbistatin), whereas in Jurkats actin flow was largely dependent upon actin nucleation. These results suggest differences in the formation and dynamics of supramolecular activation clusters that may affect the interpretation of data gathered from Jurkat T cells.
Original Research Article: Colin-York, H., et al. “Distinct actin cytoskeleton behaviour in primary and immortalised T-cells.” J Cell Science (2019): jcs-232322.

Insights into Itch

Written By: Abel B. Cortinas

Original Article: Lou et al. Journal of Immunology 2017
The Gist of It:
The sensation of itch is something that all of us are familiar with, and it can span from something as a small as the itch from a mosquito bite all the way to the itch from a medical condition such as eczema. Scientifically speaking, the biological mechanisms of extreme, chronic forms of itch are poorly understood. Part of that mystery is how relatively little is known about itch sensory nerves in the skin and how they interact with specific components of the nervous, immune, and skin systems. One example of a condition that causes itch that is poorly understood is atopic dermatitis; this is a chronic form of eczema affecting almost 1 in 5 people around the world that is characterized by extreme dryness and itching and currently has no cure. Previous studies by different groups of scientists have recently shown a correlation between human patients having atopic dermatitis, their disease severity, and the amount of a specific protein found in their cells, specifically interleukin-22 (also known as IL-22). Researchers at the Yale University School of Medicine recently studied the role of IL-22 in both mice and humans to gain a better understanding of how exactly this protein can be involved in itch, and their experiments resulted in a number of interesting findings. For example, when exposed to the house dust mite allergen, otherwise healthy mice developed atopic dermatitis and also experienced an increase in amount of IL-22 they made. Additional evidence that linked IL-22 to itch was also found when the researchers used mice that had IL-22 selectively expressed on their skin in order to cause symptoms very characteristic of atopic dermatitis. In that same mouse model, it was found that IL-22 was directly involved in increasing the amount of other specific proteins in skin cells. For example, more of the itch-inducing protein gastrin-releasing peptide was found; more of this protein was also found in the skin cells of human patients with atopic dermatitis. Overall, these findings helped indicate that IL-22 plays an important role in the initiation and development of atopic dermatitis and, more broadly, in the sensation of itch. Understanding what causes the itching sensation can help us find ways to stop it to improve the lives of the many people with conditions that cause itch.
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The Nitty Gritty:
In a house dust mite (HDM) allergen-induced atopic dermatitis model, Lou et al. found that IL-22 as well as Th2 cytokine expression was significantly increased in the lesion skin of mice with atopic dermatitis. Additionally, a K5-tTA and TRE-Tight-IL-22 skin-specific inducible transgenic system was utilized in live mice to analyze the effects of IL-22 in a controlled fashion. It was found that, when selectively expressed in the skin, IL-22 caused chronic pruritic dermatitis with many key clinical, immunopathological, and molecular features resembling those in human patients with atopic dermatitis. These clinical features included chronic pruritis, skin barrier impairment, local and system-biased Th2 responses, increased susceptibilities to Staphylococcus aureus colonization, and enhanced antigen-sensitization, highlighting IL-22 as an important link between the skin barrier and adaptive immunity. Furthermore, not only did IL-22 transgenic mice exhibit enhanced dermatitis upon epidermal allergen exposure, but the IL-22 mice with chronic pruritus also had robust expression of gastrin-releasing peptide (GRP) in dermal afferents and skin-innervating dorsal root ganglia (DRG). To corroborate the translational evidence of these findings in mice, it was observed that IL-22 directly upregulated the expression of GRP and the GRP receptor in human primary keratinocytes, and GRP synergistically enhanced IL-22–induced epithelial-keratinocyte innate type 2 cytokines. Also, highly increased expression of GRP in dermal cells and dermal afferent nerves in human atopic dermatitis lesions compared with GRP expression in normal skin were observed.
Original Research Article: Lou, H., et al. “Expression of IL-22 in the skin causes Th2-biased immunity, epidermal barrier dysfunction, and pruritus via stimulating epithelial Th2 cytokines and the GRP pathway.J Immunol 198.7 (2017): 2543-2555.