To our knowledge, the current study was the first one to investigate the diagnostic value of GSDMD in differential diagnosing different kinds of PE (including pleural transudate, TPE, MPE, and PPE). Our results indicate that the concentration of GSDMD not only acts as a novel biomarker for differential diagnosis of pleural fluid with a high diagnostic sensitivity and disease progression prediction but also can combine with ADA and LDH to improve the diagnostic ability. Moreover, PE-GSDMD displayed drug efficacy monitoring. Additionally, we found GSDMD in PE was secreted by NCs, manifested by the correlation analysis and cell infection results.
Pyroptosis is a form of pathogen-induced and caspase-dependent cell death type characterized by the exposure of phosphatidylserine, pore-formation, cell membrane rupture and secretion of cytoplasmic contents, including IL-1β, LDH and IL-18 [16]. Recent studies show that pyroptosis can be triggered by diverse pathological stimuli such as infectious disease, nervous system diseases and cancer. When the body is infected with bacteria, lipopolysaccharide (LPS) will combine caspase-4, caspase-5, and caspase-11, acting as cytosolic LPS receptors and induce cell pyroptosis [17, 18]. Similarly, through pyroptosis macrophage infected with Mtb can destroy the survival environment of Mtb, and eventually prevent Mtb replication [19, 20]. Mtb activated the canonical NLRP3 inflammasome by inducing potassium efflux upon infection of monocytes and macrophages, followed by GSDMD-dependent pyroptosis [21]. NLRP3 and AIM2 are considered two crucial kinds of pattern recognition receptors (PRR) which can activate cell pyroptosis after recognizing pathogen-associated molecular patterns (PAMP) [22, 23]. According to a large number of reports, microRNA induction [24], chemotherapy drugs [25], LXRs receptor mediation [26] and other manners can induce the occurrence of pyroptosis of tumor cells. Thereinto, microRNA, approximately 21-23 nucleotides in length, can directly target caspase-1, thereby inhibiting tumor cell proliferation and migration [24].
A recent report displays that mediated by ESCRT III components, a calcium-dependent membrane repair machinery, can antagonize the execution phase of pyroptosis [9]. Calcium flux is an evolutionarily conserved trigger for cell membrane repair through exocytosis of vesicles such as lysosomes, mobilization of annexins, and recruitment of ESCRT machinery to sites of membrane injury [27]. Based on this membrane repair machinery, cell-free culture supernatants will detect GSDMD, as has been reported [28]. What’s more, what could be detected in culture supernatants is GSDMD-NT, and GSDMD was only detected in the cell. Additionally, pyroptosis is a kind of cell lytic death way. After membrane rupture, GSDMD releases in large quantities. These are the reasons that GSDMD can be detected in pleural suffusion.
Biochemical testing of body fluid has been widely performed in the differential diagnosis between transudate and pathological pleural effusions. Though traditional biomarkers, such as ADA, LDH, glucose, protein, have been applied and new markers like IL-27 [29], IFN-γ [30], CXCL9 and CXCL11 [31] are been developing, ADA is still the most cost-effective pleural fluid marker. However, its value is questioned in antidiastole and a low prevalence setting. We have reported previously using proteome analysis for antidiastole of TPE and MPE [32]. In this study, we innovatively developed GSDMD, an indispensable molecular requirement in pyroptosis, as a novel biomarker for PE-related disease diagnosis and antidiastole of PE. GSDMD showed higher diagnostic accuracy than ADA and LDH in distinguishing transudate and other pathological pleural effusions, with 0.973 average level of the AUC for GSDMD, 0.868 for ADA, and 0.815 for LDH. It is worth to mentioning that the sensitivity and specificity for distinguishing transudate from TPE were 100% and 97.53% (AUC = 0.990), at the cut-off value of 18.40 ng/mL. What’s more, GSDMD not only manifests predominant advantage in the antidiastole of TPE but also performs well in the antidiastole of TPE and PPE (AUC = 0.885), and TPE and MPE (AUC=0.848). To make better clinical practical performance, we also calculated the cut-off value for GSDMD was selected based on the highest diagnostic accuracy. Although there existed some deficiency of GSDMD in the differential diagnosis of pleural, such as the antidiastole of MPE and PPE, and TPE and PPE, it was indisputable that GSDMD had a good diagnose performance. Furthermore, the combination of the three indexes further improved the diagnostic ability.
As we mentioned before, GSDMD can be detected in PE as a sign of pyroptosis in theory. And interestingly there existed an arresting positive correlation between the concentration of GSDMD and the count of NCs, which suggested GSDMD might be secreted by these pleural local NCs. After NCs infected by bacteria, the level of GSDMD in culture supernatants presented a growth trend in a time-dependent manner. Besides, with the extension of incubation time, a notable increase in IL-1β and LDH, as the main feature of pyroptosis [33], could be observed. These are consistent with the previous findings that pyroptosis mainly occurs in monocytes, macrophages, and also nuetrophils [34-35], to induce lytic cell death, release bacteria and expose them to reactive oxygen species (ROS) in neutrophils [36]. These data, on the one hand, confirmed a large number of GSDMD would be generated and secreted by NCs, on the other, after infection secreted-GSDMD released apace and also increased with time, which made it an ideal Inflammatory marker. Moreover, the invariability of the level of intracellular GSDMD after one hour may result of gasdermin pores have formed and cytoplasmic contents outflowed consistently.
The pore in cell membrane formed by gasdermin-N domains will elicit pyroptosis, but also generate hyperactive cells which possess lower amounts of GSDMD pores than pyroptotic cells [37]. During pyroptosis, cell membrane ruptured and cytosolic protein released, and the same with hyperactive cells. For instance, it has been confirmed that the pore-forming protein gasdermin D regulates IL-1 release from hyperactive macrophages [37]. It hints that GSDMD may become a high sensitivity index than other inflammatory biomarkers.Compared with LDH and IFN-γ, also acting as inflammatory biomarkers [38, 39], at the presence of E.coil, S.aureus and Salmonella, GSDMD displayed the most rapid and largest degree of growth tendency of GSDMD in the culture supernatant. In the previous report, after 24h the concentration of IL-27 in the mononuclear cells culture supernatants at the present presence of Mtb antigen began to increase [40]. But the concentration of GSDMD which began to increase after 1h changed faster and could make a contribution to early diagnosis. These showed that GSDMD diagnosed infectious pneumonia with a higher sensitivity. Expressed by innate immune cells, IL-1β has been demonstrated to play a role in many inflammatory diseases, as well as in different cancers [41]. The higher diagnostic sensitivity at the presence of PA might lie in the following reason. The pseudomonas aeruginosa mannose sensitive haemagglutination strain (PA-MSHA)-primed dendritic cells (DCs) directed T cell differentiation and primed Th cell to Th1 by elevated secretion of IFN-γ [42]. In conclusion, under various kinds of pathologic status especially in the case of bacteria infection, the level of GSDMD changed quick and large variation amplitude, which improved the diagnostic sensitivity and the ability of early diagnosis. Meanwhile, for regulating the release of the proinflammatory cytokines IL-1β and IL-18 and pyroptotic cell death, GSDMD would possess higher diagnostic specificity. Besides, GSDMD had an advantage in diagnostic specificity in bacteria infectious which was the main cause of pyroptosis.
We further examined the change in PE-GSDMD during bacterial pneumonia treatment with an antibiotic. The responder was gradually decreased from baseline during therapy, while nonresponders showed the opposite tendency in PE-GSDMD. The research revealed % of GSDMD change at day 4 could predict the treatment response at an early stage (AUC = 0.945, Specificity = 96.15%) and be better than other traditional indicators. The utility of procalcitonin (PCT)-guided antibiotic treatment of intensive care patients has been postulated [43]. There is research reported that the AUC of PCT clearance rate at 5 and 9 days were 0.648 and 0.729 on the therapeutic effect of ventilator-associated pneumonia [44]. As one of the important causes of toxic and side effects of some chemotherapy drugs, caspase-3 activation can trigger necrosis by cleaving GSDME [45]. This means that detection based on GSDME expression level can be used as a better tool for prognosis interpretation. Besides, it’s difficult to repeatedly extract PE from lung cancer patients. There are some limitations to our study. Firstly, the limited population was enrolled in this study which may lead to bias. Also, it’s difficult to rule out the misclassification of pleural fluid, especially the transudate group and PPE effusion and the collection of PE is also an invasive test. Moreover, further study needs to be conducted to clarify the normal level of serum GSDMD.