Title : Ongoing journey – targeting citrullinated histone H3 (CitH3) for early diagnosis and treatment of sepsis
Abstract:
Over the past decade, our research team has dedicated our efforts towards studying protein post-translational modifications with the goal of early diagnosis and treatment of sepsis. Our comprehensive and intensive research has culminated in the publication of approximately 40 manuscripts on the topic (click to see our sepsis project).
Citrullination of histone H3 by nuclear-localized peptidylarginine deiminase (PAD) is recognized as an early phase in a distinct form of cell death, known as "neutrophil extracellular traps" or NETosis. Histone H3 citrullination acts as a convergence point for a variety of inflammatory signals, triggering the neutrophil response to infections. Hyper-citrullinated histone H3 (CitH3) is detected in immune cells and the bloodstream during sepsis (infectious), but not subsequent hemorrhagic shock (non-infectious), according to rodent models and human studies (our preliminary data). Furthermore, septic mice treated with a pan-PAD inhibitor or an anti-CitH3 antibody exhibit a significant improvement in survival, thereby illuminating the pathway for the proposed clinical discovery study.
Our team initially pinpointed CitH3 as a potential serum protein biomarker in a lethal mouse model subjected to lipopolysaccharide (LPS)-induced shock in 2011. Since then, we have persevered in this research direction, generating substantial data that reinforces the notion of CitH3 acting not only as a biomarker but also as a contributing factor for endotoxic/septic shock and associated organ damage.
Over the last decade, we have tackled a substantial concern regarding the use of commercial anti-CitH3 antibodies in research. A range of studies highlighted the limitation of commercially available CitH3 monoclonal antibody (mAb) (with the epitope of 3 Cit at R2/R8/R17) in accurately quantifying serum CitH3 levels. This antibody has been employed to link CitH3 with advanced cancer patients displaying an intensified inflammatory response, suggesting its potential as a diagnostic and prognostic blood marker. Nonetheless, the conflicting findings reported in these studies are predominantly due to the limitations inherent in the commercial antibody.
One significant contribution from our lab is the development of a novel anti-CitH3 antibody, which contains an epitope of 4 Cit at R2/R8/R17/R26 and offers a more precise diagnosis and prognosis of sepsis. This CitH3 mAb (4 Cit) recognizes epitopes on CitH3 generated by both PAD4 (R2/R8/R17) and PAD2 (R26). Our team has proven that this antibody can enhance survival rates in a mouse sepsis model. Upon evaluating the specificity and sensitivity of our CitH3 mAb (4 Cit), we found it to be superior to the commercial CitH3 mAb (3 Cit) in specificity and sensitivity. Our antibody binds to all circulating CitH3 produced by PAD2 and PAD4, thereby addressing the deficiency of commercial CitH3 antibodies. In fact, our antibody fills the void identified by Neeli and Radic in their research on citrullinated histone antibodies (Current Challenges and Limitations in Antibody-Based Detection of Citrullinated Histones, Frontiers in Immunology, 2016). Utilizing an assay based on our CitH3 mAb (4 Cit), we established that CitH3 is a dependable blood biomarker for the diagnosis and treatment of endotoxic shock (Pan et al. Scientific Reports 7: 8972 PMID: 28827548; Deng et al. Frontiers in Immunology. 2020;10: 2957. PMID: 32943500). Other scientists have noted in Commentary that our findings could facilitate early detection and monitoring sepsis progression.
We've created a sandwich enzyme-linked immunosorbent assay (ELISA) utilizing our novel CitH3 mAb as the capture antibody. This ELISA shows greater sensitivity in comparison to commercially available ELISA kits. Utilizing our kit, we've recently shown that CitH3 levels can effectively distinguish between patients with septic and non-septic shock, as well as correlate with the severity of the disease. These recent findings have been published in Infection.
The University of Michigan (UM) holds the patent for this innovative CitH3-ELISA kit and is actively collaborating with a pharmaceutical company to bring it to clinical use. The successful commercialization of this kit will significantly benefit the University. Concurrently, we have partnered with Dr. Katsuo Kurabayashi's team from the Department of Mechanical and Aerospace Engineering at NYU Tandon School of Engineering. Together, we have engineered an integrated plasmo-photoelectronic biosensor for the rapid detection (within 15 minutes) of the CitH3 biomarker. This biosensor aids in the prompt diagnosis of sepsis in mice, as discussed in our publication in Small, and it holds promise for clinical applications in point-of-care precision medicine.
Beyond the creation of a novel anti-CitH3 mouse monoclonal antibody (mAb), we've also developed a humanized version of the CitH3 mAb (hCitH3 mAb). Our findings indicate that the hCitH3 mAb demonstrates a significantly stronger binding affinity to CitH3 when compared with the control mAb.
Our key contributions to the literature on CitH3 as a biomarker and mediator are detailed in the following publications:
