Preventing the oxidation of cysteine thiols of TLRs maybe a treatment strategy for sepsis and hyperoxic injury

Significance 

Toll-like receptors (TLR) play an important role in the natural immunity of mammals and the host’s response to infection. TLRs possess extra-cytoplasmic domains (ectodomains) containing leucine-rich repeats (LRRs) and cysteine-rich regions which are responsible for binding to various molecules. TLR2 and TLR4 are members of the TLR family that initiate the natural immune system in response to “molecular patterns” associated with pathogens expressed by different infectious microorganisms. For example, TLR4 is the receptor for lipopolysaccharide (LPS), a molecule found in the cell wall of Gram-negative bacteria, which causes septic shock through overwhelming systemic inflammatory reactions.

It has been shown that the binding of TLR4 to its co-receptor – MD2 and the binding of bacterial LPS to this receptor complex is fostered by the cysteine thiols of the TLR4. These cysteine thiols are susceptible to oxidation, but previous studies have only examined their oxidation state in reference to TLR oligomerization. Understanding the mechanism and control of TLR signaling has been instrumental in formulating medications to treat various infections like sepsis and inflammatory diseases. Despite all the progress made in the treatment of infections, the increase in the number of new pathogens, antibiotic-resistant strains and immunosuppressed patients all pose serious therapeutic challenges.

Dr. Zsuzsanna K. Zsengellér and Dr. Norma P. Gerard from the Harvard Medical center proposed a hypothesis that the oxidation state of cysteine thiols on the ectodomain of TLR2 and TLR4 are critical for both pathogen- and hyperoxia-initiated intracellular  signaling. Their research goal was to determine if modification of the free thiols on the ectodomain of TLR2 and TLR4 would modulate receptor signaling in response to these stimuli. Their findings confirmed their hypothesis as it showed that indeed the free thiol groups on the TLR ectodomain undergo oxidation when exposed to pathogen or hyperoxia. The work is now published in the Journal of Immunobiology.

The researchers carried out sequence analysis of various proteins and found that there were homologous cysteines in the Leucine-Rich Repeat C-terminal (LRRCT) region of TLR ectodomains such as human TLR 1, 2, 3, 4 and Drosophila Toll.  In addition, they conducted in vitro experiments by exposing human HEK293 cells expressing either TLR2 or 4 as well as CD14+MD2, hamster CHO-3E10 cells and isolated mouse peritoneal macrophages to bacterial LPS, or yeast wall zymosan or hyperoxia. The authors found that pre-incubation of the cells with thiol-modifying compounds before their exposure to LPS or zymosan, resulted in a blockade of intracellular signaling from TLR4 and TLR2. These studies may provide clues for the development of novel treatment interventions for yeast infection, sepsis and hyperoxia-induced cell injury.

In summary, the study by Zsengellér and Gerard confirmed that there are free thiol groups on the ectodomain TLR protein and they undergo oxidation upon exposure to pathogen or hyperoxia. The blockade of this oxidation reduced the inflammatory responses of the cells caused by these stimuli. The study advanced our knowledge on the mechanism and control of signaling from TLRs. The authors recommend future studies to examine and identify the role of the TLR ectodomain cysteines in activating intracellular signaling.

Preventing the oxidation of cysteine thiols of TLRs maybe a treatment strategy for sepsis and hyperoxic injury - Medicine Innovates
Mechanism of thiol oxidation upon hyperoxia injury and possible modification of TLR cysteines. TLR’s contain several cysteines on their ectodomain. In control condition such as normoxia (A) the TLRs have free cysteine thiols that can be demonstrated by binding of alkylating agent Alexa-488 maleimide to the ectodomain of TLR4. Upon hyperoxia exposure the thiols are oxidized and cannot bind Alexa-488 maleimide, thus oxidation of thiols is reflected in decreased measurement of Alexa-488 maleimide by Western blot. The possible alterations of thiols under hyperoxia can be, (B) intramolecular disulfide bond formation, (C) homo or heterodimerization with TLRs, (D) or TLR thiols dimerize with other cysteines thiols of a different molecule such as MD2. (Botos, I., D. M. Segal, and D. R. Davies. 2011. The structural biology of Toll-like receptors. Structure 19: 447-459). The theoretical changes may induce receptor oligomerization, or conformational changes of the receptor protein which leads to alteration in intracellular signaling. (E) a general chemical reaction of a thiol with maleimide (Thermo Fisher Scientific # A10254 Alexa Fluor™ 488 C5 Maleimide). We demonstrated, that pretreatment of various cell lines with the thiol-alkylating agent before hyperoxia or pathogen challenge resulted in decreased intracellular signaling, suggesting that oxidation of the free thiols is critical for TLR signaling. The free cysteine thiols on TLRs could be a useful target for anti-inflammatory agents designed to prevent pathogen or hyperoxia- mediated TLR activation.

About the author

Zsuzsanna K. Zsengellér was born in Szeged, Hungary. She graduated from the Albert Szent-Györgyi Medical University with M.D. / Ph.D. (1998) degrees in medical sciences under the supervision of Prof. Aranka Laszló and Prof. Ernõ Duda. She started her residency in the Department of Pediatrics at Albert Szent-Györgyi Medical University. Following her residency she was invited for a postdoctoral positions with Jeffrey Whitsett at the Department of Pulmonary Biology, Children’s Hospital Medical Center Cincinnati, USA. In 2000 she joined Prof. Norma Gerard’s research group at Children’s Hospital Boston, and became a faculty in 2005 at Harvard Medical School.

Her main interest was to explore Toll-like receptor signaling pathways in lung inflammation and injury. She also worked in the biotechnology industry for 3 years as Director of Investigational Pathology (Radikal Therapeutics, Beverly MA). In 2009, she was promoted to Instructor of Medicine at Harvard Medical School and joined the group of Dr. Ananth Karumanchi at Beth Israel Deaconess Medical Center studying preeclampsia pathogenesis and treatment strategies. She has mentored several graduate and post-graduate students at BIDMC and believes that the understanding of pathways of disease will be fundamental to the development of novel therapeutics. Membership of Academic Societies: The American Society of Investigational Pathology, The Histochemical Society, The American Heart Association, The American Society of Nephrology. She has been awarded from the NIH and The American Heart Association.

Reference

Zsuzsanna K. Zsengellér, Norma P. Gerard.  The oxidation state of cysteine thiols on the ectodomain of TLR2 and TLR4 influences intracellular signaling. Immunobiology. 2020 Mar;225(2):151895. doi: 10.1016/j.imbio.2019.12.004. Epub 2019 Dec 5  PMID: 31843260.

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