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

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.