A multidisciplinary team of investigators at Vanderbilt University Medical Center has discovered that blocking an iron uptake receptor reduces disease pathology and promotes the activity of anti-inflammatory regulatory T cells in a mouse model of SLE. The findings were published Jan. 13 in the journal Science Immunology.
Treatments for lupus aim to control symptoms, reduce immune system attack of tissues, and protect organs from damage. Only one targeted biologic agent has been approved for treating SLE, belimumab in 2011.
It has been a real challenge to come up with new therapies for lupus. The patient population and the disease are heterogeneous, which makes it difficult to design and conduct clinical trials.”
Jeffrey Rathmell, PhD, Professor of Pathology, Microbiology and Immunology and Cornelius Vanderbilt Chair in Immunobiology
Rathmell’s group has had a long-standing interest in lupus as part of a broader effort to understand mechanisms of autoimmunity.
When postdoctoral fellow Kelsey Voss, PhD, began studying T cell metabolism in lupus, she noticed that iron appeared to be a “common denominator in many of the problems in T cells,” she said. She was also intrigued by the finding that T cells from patients with lupus have high iron levels, even though patients are often anemic.
“It was not clear why the T cells were high in iron, or what that meant,” said Voss, first author of the Science Immunology paper.
To explore T cell iron metabolism in lupus, Voss and Rathmell drew on the expertise of other investigators at VUMC:
- Eric Skaar, PhD, and his team are experienced in the study of iron and other metals;
- Amy Major, PhD, and her group provided a mouse model of SLE; and
- Michelle Ormseth, MD, MSCI, and her team recruited patients with SLE to provide blood samples.
First, Voss used a CRISPR genome editing screen to evaluate iron-handling genes in T cells. She identified the transferrin receptor, which imports iron into cells, as critical for inflammatory T cells and inhibitory for anti-inflammatory regulatory T cells.
“We see a lot of complications coming from that -; the mitochondria don’t function properly, and other signaling pathways are altered,” Voss said.
An antibody that blocks the transferrin receptor reduced intracellular iron levels, inhibited inflammatory T cell activity, and enhanced regulatory T cell activity. Treatment of SLE-prone mice with the antibody reduced kidney and liver pathology and increased production of the anti-inflammatory factor, IL-10.
“It was really surprising and exciting to find different effects of the transferrin receptor in different types of T cells,” Voss said. “If you’re trying to target an autoimmune disease by affecting T cell function, you want to inhibit inflammatory T cells but not harm regulatory T cells. That’s exactly what targeting the transferrin receptor did.”
The researchers are interested in developing transferrin receptor antibodies that bind specifically to T cells, to avoid any potential off-target effects (the transferrin receptor mediates iron uptake in many cell types). They are also interested in studying the details of their unexpected discovery that blocking the transferrin receptor enhances regulatory T cell activity.
Skaar is the Ernest W. Goodpasture Professor of Pathology and director of the Vanderbilt Institute for Infection, Immunology, and Inflammation. Major, associate professor of Medicine, and Ormseth, assistant professor of Medicine, are faculty members in the Division of Rheumatology and Immunology. Rathmell is the director of the Vanderbilt Center for Immunobiology.
Other authors of the study include Allison Sewell, Evan Krystofiak, PhD, Katherine Gibson-Corley, DVM, PhD, Arissa Young, MD, Jacob Basham, MD, Ayaka Sugiura, PhD, Emily Arner, PhD, William Beavers, PhD, Dillon Kunkle, PhD, Megan Dickson, Gabriel Needle, and W. Kimryn Rathmell, MD, PhD.
The research was supported by the National Institutes of Health (grants DK105550, AI153167, DK101003, AI150701, CA253718) and the Lupus Research Alliance William Paul Distinguished Innovator Award to Jeffrey Rathmell.
Iron is one of the most abundant elements on Earth and essential to almost all organisms.
Iron exists in a wide range of oxidation states, −2 to +7.
Chemically, the most common and biologically relevant oxidation states of iron are +2 and +3, ferrous (Fe2+) and ferric (Fe3+) iron.
Ferrous (Fe2+) iron is more soluble and bioavailable than its ferric (Fe3+) form and that the interchangeability of these two ionic forms of iron via oxidation/reduction are essential for the function of many cellular proteins.
Levels of iron in the body are strictly controlled through finely tuned complex mechanisms, to prevent the cytotoxicity that is induced by accumulation of this metal and to allow physiologically tolerable iron levels to serve as a critical catalytic component of many proteins and enzymes, called metalloproteins.
Metalloproteins can directly bind iron or use iron-containing complexes such as heme or iron-sulfur (Fe-S) clusters. Such proteins have diverse and essential processes within the cell, including oxygen carrying (hemoglobin), oxygen storage (myoglobin), energy production (cytochrome-C), cellular metabolism (amino acid oxidases, fatty acid desaturases), detoxification (cytochrome P450, catalase), and host defense (myeloperoxidase, nitric oxide synthase, IDO, NAPH oxidase) (Muckenthaler et al., 2017).
Although the chemistry of iron will not be discussed here in detail, Fenton/Haber-Weiss chemistry is a very important reaction with widespread effects on biological systems under normal and pathophysiological conditions: ferrous (Fe2+) iron reacts with hydrogen peroxide to form the hydroxyl ion (OH−), the hydroxyl radical (OH•) and ferric (Fe3+) iron (Koskenkorva-Frank et al., 2013). The OH• radical is a non-selective, highly toxic oxidant.
As mitochondria produce ATP by oxidative phosphorylation (OXPHOS), reactive oxygen species (ROS) by-products such as superoxide are generated from the electron transport chain (ETC). Superoxide radicals can reduce and liberate Fe3+ from ferritin or liberate Fe2+ from Fe-S clusters (see below). Biologically-available iron not sequestered is thus a dangerous source of damaging radicals (Breuer et al., 2008). It is important to note that not all free radicals are detrimental and not all antioxidants are beneficial.
Normal physiology is a balance between the two: antioxidants maintain levels of ROS that permit them to perform useful biological functions, such as neutrophil-mediated killing of phagocytosed bacteria or enhanced T cell proliferation after TCR stimulation, while minimizing by-stander damage. However, under pathophysiological conditions, such as enhanced mitochondrial stress, this balance gets perturbed to the detriment of the organism.
Iron is essential for many physiological processes in the body including erythropoiesis, immune function and host defense, as well as essential cellular activities such as DNA replication and repair, mitochondrial function including OXPHOS and enzymatic reactions which require iron as a cofactor.
Extensive research by many groups has unveiled the regulatory network governing iron homeostasis in the body and inside the cell, as well as the links between disturbances of iron homeostasis and disease. Iron deficiency is the most common pathology of iron homeostasis, eventually resulting in iron deficiency anemia, the most frequent anemia worldwide (Camaschella, 2015).
The second most frequent anemia, anemia of inflammation (also called anemia of chronic disease), largely results from inflammation-driven retention of iron in certain immune cells, resulting in iron-limited erythropoiesis (Weiss et al., 2019). This latter pathology reflects the complex regulatory interactions between iron and the immune system, which emerged evolutionary from a strategy of the organism to withhold nutrient iron from invading pathogens, a defense mechanism known as nutritional immunity.
Accordingly, iron trafficking is controlled by cytokines and acute phase proteins, whereas the metal itself promotes lymphocyte and macrophage differentiation, anti-microbial immune effector function, and immune cell metabolism, as we will discuss later (Ganz and Nemeth, 2015; Soares and Weiss, 2015). Thus, imbalances in iron homeostasis are prevalent in infections, cancer as well as autoimmunity, and its pathophysiological or therapeutic modulation impacts on the outcome of such diseases.
Evolution reveals its mastery in the way the body and immune cells strike a balance between iron supply and demand with pathways tightly regulating iron levels extra- and intra-cellularly, from its uptake, use, storage, and export, collectively referred to as the iron cycle (Figure 1, Table 1). In the next sections we will describe the various stages of the iron cycle and give an overview of how iron levels are monitored and regulated inside the cell. This cellular regulation of iron is applicable to practically every cell in the body, including all immune cells.
Iron metabolism in the cell. Intracellular iron levels are strictly controlled as too little or too much can be detrimental to the health of the cell. Therefore, (1)-iron uptake, (2)-utilization, (3)-storage and (4)-export need to be managed in a coordinated manner, as well as the conversion between the oxidation states of iron (Fe2+ and Fe3+) in the cell. (1) Iron-bound transferrin (TF-Fe3+) and NTBI (non-transferrin-bound iron) are taken up into the cell by the iron importers DMT1 and ZIP14. STEAP3 is a ferrireductase which reduces Fe3+ to Fe2+, which can then be imported. (2) Once inside the cell, the bioavailable and more soluble Fe2+ is used for various biological processes– DNA replication, ROS production via Fenton/Haber-Weiss (F/H-W) chemistry, mitochondrial bioenergetics, Fe-S and heme biosynthesis, as well as a plethora of proteins which utilize the metal to carry out their functions. (3) Excess Fe2+ iron is dangerous due to its role in ROS production. Therefore, it needs to be stored but, at the same time, be readily available for use. This is achieved by a particular arrangement of ferritin proteins designated the “ferritin cage” which stores the more inert, insoluble Fe3+ form of iron. When intracellular levels are low, this ferritin cage is signaled for destruction by NCOA4 thus releasing the stored iron. (4) If intracellular iron levels are saturated, then the iron must be exported out of the cell. This is achieved by the iron exporter ferroportin (FPN). Once outside the cell the Fe2+ iron is oxidized to Fe3+ (via CP, HEPH, HEPHL). (5) Finally, Fe3+ iron is then bound to transferrin (Tf-Fe3+) and enters the circulation to begin the cycle again. Notably, hepcidin is an iron-controlling hormone produced by the liver. When systemic iron levels are high in the blood, hepcidin is produced and leads to the degradation of FPN on cells thus preventing cellular release of iron into the blood. Conversely, when iron blood levels are low, hepcidin expression is reduced.
Table 1
The major players of the Iron Cycle.
| Process | Protein | Location | Function |
|---|---|---|---|
| Iron intestinal uptake | DcytB DMT1 | Gut lumen > enterocyte Gut lumen > enterocyte | Ferrireductase (reduces Fe3+ to Fe2+) iron transporter of Fe2+ |
| Unidentified HO1 HO2 | Gut lumen > enterocyte inside enterocyte | Heme-conjugated iron Breaks down the heme to produce free Fe2+ | |
| PCBP2 | Inside enterocyte | Chaperones Fe2+ to basolateral side of enterocyte | |
| Release of dietary iron to circulation | FPN Hephaestin | Enterocyte > circulation | Fe2+ exporter from enterocyte Ferroxidase (oxidizes Fe2+ to Fe3+) |
| In the circulation | TF NTBI | In the blood In the blood | TF binds and transports Fe3+ (TF-Fe3+ complex) Non-transferrin bound iron |
| Cellular iron uptake | TFR1 Low pH STEAP3 DMT1 | Cell surface Endosome Endosome Endosome > cytosol | Binds and endocytoses TF-Fe3+ Release of Fe3+ from TF-Fe3+ (TFR1 recycled to surface) Ferrireductase (reduces Fe3+ to Fe2+) Iron transporter of Fe2+ |
| ZIP14 DMT1 | Cell surface > cytosol Cell surface > cytosol | Binds and uptakes NTBI into cell | |
| Intracellular iron storage/release | FTH1 FTL1 | Cytosol/mitochondria | Components of “ferritin cage” |
| NCOA4 | Cytosol | Targets ferritin for autosomal degradation to release iron | |
| Iron cellular export | FPN | Cytosol > circulation | Fe2+ exporter from the cell |
| CP HEPH HEPHL1 | Outer cell surface | Ferroxidase (oxidizes Fe2+ to Fe3+) |
This table depicts the various stages of the iron cycle, the proteins involved at each stage as well as their function, and the location-of-action of these proteins.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883604/
More information: Kelsey Voss et al, Elevated transferrin receptor impairs T cell metabolism and function in systemic lupus erythematosus, Science Immunology (2023). DOI: 10.1126/sciimmunol.abq0178. www.science.org/doi/10.1126/sciimmunol.abq0178
