Much research in the past decade has been focused on the exploration of one or a panel of inflammatory mediators for the diagnosis of systemic infection. ¹ All these studies used the candidate biomarkers approach for selecting specific mediators in the inflammatory cascade or cell surface antigens that are either up- or down-regulated in response to pathogen invasion. This approach, however, has the disadvantage of limiting the search of potential biomarkers to only known proteins. Mass spectrometry-based proteomic profiling technologies, such as isobaric tag for relative and absolute quantitation (iTRAQ), have been used to identify host-response proteins for diagnosis of many diseases and pathologic conditions. Using iTRAQ technology, we have recently found that LPS-induced sepsis causes the rise of a panel of proteins in serum, including some histone proteins (un-published data).

Posttranslational modification of a histone protein, such as deimination (citrullination) and acetylation, can change its structure and function. Deimination/citrullination of histones by peptidyl arginine deiminase 4 (PAD4) can be induced by lipopolysaccharide (LPS). PAD4 is an enzyme previously known to convert protein arginine (Arg) to citrulline (Cit), a nonconventional amino acid in proteins. ² This enzyme was first identified in human HL-60 leukemia cells upon differentiation along the granulocyte lineage ³ and is highly expressed in peripheral blood neutrophils. ⁴ It is known that the LPS-induced histone deimination/citrullination is an early response to inflammatory stimuli in neutrophils. ⁵ Moreover, citrullinated histone H3 (Cit H3) has been identified as a component of neutrophil extracellular traps (NETs) that are produced by degranulating neutrophils. This Cit H3 is released in the extracellular space as part of the neutrophiil response to infection. ⁶

The accumulated data point towards a link between citrullinated proteins and pathogenesis of diseases such as rheumatoid arthritis, multiple sclerosis and psoriasis. Lundberg et al showed that appearance and amounts of citrullinated proteins in arthritic joints of experimental animals are correlated the severity of inflammation. ⁷ However, these proteins reflect their relationship with autoimmune diseases. It remains unknown whether a citrullinated protein in neutrophils could serve as a serum biomarker to predict the severity of sepsis.

Our studies also suggest some possible answers to other questions such as where the serum histones come from, how they are released into the blood stream, why an early rise in the circulating Cit H3 associates with survival in this model of shock, and whether Cit H3 is more useful than conventional biomarker/mediator(s). Some of these issues are discussed in the following paragraphs.

Our findings show that alteration in serum Cit H3 is associated with H3 in vivo when animals are injected with large dose of LPS (Fig 3A). Also, it seems that these two proteins are secreted together into the medium from the HL-60 cells in vitro (Fig 2C). It is not clear where the histone proteins come from during sepsis. ^{16, 17} However, neutrophils are an attractive possibility, as they are the most abundant white blood cells and they are actively involved in post-septic immune response. ¹⁶ NETs formation is a conceivable action for neutrophils to release the histone proteins into the blood circulation. Conventionally, two strategies are considered for neutrophils to contain and clear bacterial infection: phagocytosis and degranulation. Recently, a third strategy has been proposed, i.e., formation of NETs. ¹⁸ NETs arise from the release of neutrophil nuclear contents into the extracellular space. They are composed of decondensed chromatin that is decorated with granular as well as cytoplasmic proteins. Neutrophils use these three strategies to combat and clear microbes, but they operate over different timescales. Phogocytosis takes about 10 min, degranulation releases antimicrobial molecules in 30 min, and NETs usually need 2-3 h to secrete the nuclear proteins. ¹⁹ In our studies, we did not find H3 and Cit H3 in the extracellular space (serum and the cultured cell medium) until about 3 h (data not shown), suggesting that NETs may be the major origin of the histone proteins.

Many researchers have found that histone deimination/citrullination is an important molecular mechanism for NET formation. ¹⁹ Experiments performed on the netrophilic HL-60 cell line also suggest that NET formation depends on histone deimination/citrullination, which is catalyzed by PAD4. ¹² Neutrophils express high levels of PAD4 enzyme. ⁴ In response to various stimuli, including pathogen infection and inflammatory response, PAD4 in neutrophils rapidly hydrolyzes quanidino group of histones to ureido group and ammonia so that histones become deiminated/citrullinated histones. These mediate chromatin decondensation and NET formation. Using the HL-60 granulocytes as a model system, Wang et al. have recently shown that activated PAD4 and deiminated/citrullinated histone initiate chromatin decondensation, and that global histone hypercitrullination regulates the unfolding of chromatin structures during NET formation. ¹² Neeli et al. found that histone deimination in neutrophils represents a rapid and robust reaction to signals arising in a microbial infection or inflammatory stimuli. ⁵ This data, together with our current findings, clearly support the concept that histone deimination/citrullination plays a role in the generation of the circulating Cit H3 and H3.

To date, two models describing the release of NETs have been proposed: a DNA extrusion mechanism from intact cells, and a cell death mechanism (Fig 5). Addressing the question of how neutrophils form NETs, Fuchs et al. further monitored the individual cells via live video microscopy which confirmed NET formation. ²⁰ In these experiments, they demonstrated that ex vivo, activated neutrophils enter a program where the nuclear and granular membranes dissolve and the nuclear contents decondense into the cytoplasm. Then the plasma membrane ruptures and chromatin decorated with granular proteins is released into the extracellular space. This mechanism may reflect NET formation occurring upon direct stimulation by pathogens. In contrast, another study described that in the presence of bacterial LPS and platelets, neutrophils can generate NETs within minutes. ²¹ This process has been implicated in sepsis-associated microvascular thrombosis and involves indirect neutrophil stimulation mediated by platelets in the environment of slow blood flow or coagulation. ¹⁹ In our studies, we did not find an elevation in the serum histone proteins within a few minutes after LPS injection. The explanation could be that although LPS-mediated neutrophil activation needs a short time, it may not be initiated until the later stages of sepsis. At the time when we collected blood, the inflammatory process and shock were still in an early stage without any evidence of a coagulation disorder such as disseminated intravascular coagulation (DIC). With phorbol myristate acetate (PMA), Staphylococcus aureus, or Candida albicans as stimuli, Fuchs et al. reported that the entire process of NET formation takes between 2-3 hours. ²⁰ Based on their finding, we incubated the HL-60 cells with LPS for 3 h to ensure complete histone deimination and NET formation. In our in vivo experiments, we designed the time for blood collection at 3 h after LPS injection, which confirmed that sufficient/adequate time had passed for detection of serum histone proteins. In addition, from the survival experiments we observed that animals were still in good condition at 3 h after ip administration of LPS. They did not display signs of severe sepsis such as reduced mobility, conjunctivitis, diarrhea, and fur ruffling within 3 h. In the lethal LPS (LD) group, mice began to die at 16 h and all were dead within 23 h. Thus, three hours is fairly early in the time course of sepsis. The fact that serum Cit H3 was detectable as early as 3 h in animals that subsequently died suggests that this circulating protein can predict the severity of shock.

In the current study, we have compared Cit H3, histone H3 and TNF-α in terms of effectiveness as biomarkers. Histone H3 has been proposed as a biomarker in sepsis by Xu et al recently.⁸ This biomarker was detected in the circulation of baboons challenged with E. Coli, and an increase in the histone H3 levels was accompanied by the onset of renal dysfunction.⁸ In our experiments, we confirmed that the circulating H3 protein was induced by LPS, a component of the outer cell wall membrane of Gram-negative bacteria. But this did not correlate with the dose or lethality of the LPS. We observed that increase in circulating H3 was not only found in LPS (LD) group but also in LPS (SD) group. Similarly, TNF-α, a main pathogenic mediator in septic shock, was detected in the serum from both the LPS (LD) and LPS (SD) groups (p = 0.77). Given that Cit H3 was only noted in the LPS (LD) group, and that all of these animals subsequently died, it may be a better marker to discriminate between mild sepsis and severe sepsis. Moreover, our studies suggest that Cit H3 does not simply mirror changes in TNF-α, and actually appears to be more useful than TNF-α in monitoring the severity of LPS-induced shock. We also discovered that Cit H3 was not detected in mice that had undergone hemorrhagic shock (Fig. 3A). Based on our hypothesis that major source of Cit H3 is the neutrophil extracellular traps (NETs) after infection (Fig. 5), serum Cit H3 appears to be specific for severe sepsis.

This study has certain limitations that must be acknowledged. SAHA, a pan-histone deacetylase inhibitor, can induce histone acetylation. However, whether acetylation of histone H3 by SAHA interferes with deimination of histone H3 and its mechanism remain unknown. Also, it is not clear how many citrullination sites are involved in histone H3, since we used an antibody that can only detect citrullinated H3 at citrulline 2 + 8 + 17. Recently, Stensland et al performed liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis with alternating collision-induced dissociation (CID) and electron transfer dissociation (ETD) to specifically detect and characterize citrullinated peptides. ²² The combination of LC/MS/MS with CID and ETD might be a useful technique to help us to solve these problems in the future. Finally, we used LPS to induce shock in this model, which clearly does not replicate all the facets of a poly-microbial infection. However, this was a proof of concept study exploring the mechanistic aspects of the process. An ongoing clinical study has shown an increase in circulating Cit H3 levels in patients with ventilator associated pneumonia due to multiple organisms. We have also used a cecal ligation and puncture model to verify these findings.