Neonatal sepsis is the constellation of symptoms occurring an infection with bacterial, viral, or fungal (mostly yeast) microorganisms that leads to a systemic inflammatory response in neonates [27, 28]. Despite the extensive use of broad-spectrum antibiotics, ventilator management, resuscitative strategies, and improvement of nutritional support, four of every 10 neonates that develop sepsis die or experience major disability . The newborn immune system is complicated by its immaturity, however, akin to adult counterparts, immune exhaustion and dysfunction also occurred in neonates. Published studies have shown that co-inhibitory receptors, including PD-1, CTLA-4 and BTLA, are contributed to the progression of sepsis [20, 31–34]. Although these receptors were originally thought primarily to be inducers of anergy in lymphocytes, such as CD4+ T cells, increasing evidence verified that innate cell populations, including macrophages, monocytes and DCs, also appear to be induced to express co-inhibitory receptors, including BTLA, and the ligation of these receptors may have effects on them as well. BTLA has been showed to be detrimental in the bacteria clearance in mice by impairing the recruitment of innate inflammatory immune cells to the infected sites as well as inhibiting them to present antigen to adaptive immune system . However, the expression of BTLA as well as its regulation effect on DCs in neonates with sepsis remains to be elucidated.
In our study, we found the percentage of BTLA+mDCs in septic neonates was significantly higher than that in non-septic neonates. We conducted ROC curve analysis, and the percentage of BTLA+mDCs was a relatively better marker in predicting neonatal sepsis even though the predictive effect was not as strong as PCT. Yang and his colleagues confirmed that BTLA is selectively expressed in CD8α+DCs, but not CD8α−DCs to regulate the bacteria expansion in mice . But we did not further determine which subtypes of mDCs were elevated during neonatal sepsis, which would be our next study point. Complex immune reactions were developed during sepsis, which can be conceptualized as an occurrence of a pro-inflammatory along with a concomitant anti-inflammatory response and this persistent anti-inflammatory response is believed to contribute to the profound state of immune paralysis and late morbid outcome [26, 36]. Therefore, the increased expression of inhibitory molecular in the early stage of sepsis could prevent the cytokine storm from inducing organ failure . In this regard, BTLA has been shown could directly inhibit LPS responses in DCs and Mϕs, and the agonistic agents of BTLA might have therapeutic potential for LPS-induced endotoxic shock, which an overwhelming and uncontrolled immune responses would occur in the early phase of infection . However, if their expression still keeps in a high level during the immunosuppression stage, T cell anergy or immune cell deactivation would be induced and that is detrimental for the bacteria clearance. Our data provided herein demonstrated that those neonates with severe sepsis showed higher level of BTLA+mDCs than non-severe sepsis. As the extension of the days of hospitalization, the percentage of BTLA+mDCs showed a stepwise increase in severe septic neonates, whereas it began to decrease from the 4th days of hospitalization in non-severe septic neonates. We feel that our findings provided here suggested that not only did the percentage of BTLA+mDCs correlate to the severity of sepsis, but also it was valuable for prognostic evaluation of sepsis. The increased level of BTLA+mDCs is not conducive to the control of infection, in other words, the higher level of BTLA+mDCs in the late stage of sepsis would be a predictor of pejorative outcome in neonates with sepsis.
BTLA has been confirmed to be detrimental for the clearance of bacteria, including L. monocytogenes and Plasmodium, suggesting a role for in inhibiting innate phagocytic cell action [19, 35, 38]. Prior study also demonstrated that BTLA-expressing CD11c+ APCs from tuberculosis infected patient exhibit lower antigen uptake capacity than BTLA negative CD11c APCs . Consistently, our data demonstrated that BTLA+mDCs from septic neonates exhibited lower phagocytosis ability and bactericidal capacity when compared with BTLA−mDCs. In addition to impairing the antigen-uptake capacity of DCs, the occurrence of BTLA also has been found could negatively regulate the antigen-presenting capacity of innate cells to adaptive immune system, which is another disadvantage for bacteria clearance. CD8α+DCs from BTLA deficient mice are less efficient at cross-presenting Listerial antigen to CD8+ T cells resulting in less ability to induce T cells proliferation and IFN-γ production . Our previous study also found that BTLA-expressing CD11c+ APCs from tuberculosis infected patients display lower stimulatory capacity of CD3+T cells, especially CD8+T cells, because of the diminished expression of HLA-DR and lower production of IL-6. Interestingly, in this study, we found HLA-DR expression in mDCs was lower in neonates with sepsis than neonates with no sepsis. However, a much higher level expression of HLA-DR in mDCs was found in septic neonates than severe septic neonates and an increasing tendency was found as the extension of hospitalization, which keeps in a relative stable expression in severe septic neonates. Furthermore, HLA-DR expression in mDCs was negatively regulated by BTLA in neonates with sepsis but not no-sepsis. Combined with previous studies, our findings provided new evidence for the regulation effect of BTLA in the cell function of mDCs and might interpret partially why neonates with higher levels of BTLA in mDCs display severe sepsis and longer duration of hospitalization.
Cytokine plays an important role in influencing the cell function of DCs. It is verified that co-inhibitory receptors, including PD-1 and BTLA, could not only inhibiting innate cells effector function but also shifting them into anti-inflammatory phenotypes as well. The induced or secreted anti-inflammatory mediators, like IL-10, are thought to cause innate immune cells, including monocytes, macrophages and DCs, to become dysfunctional. The dysfunction includes reduced IL-1, TNF-α, and IL-6 pro-inflammatory cytokine, but increased IL-10, which are not conducive to the control of infection during sepsis. As an activator of DCs, the expression of IL-12 also was found to be reduced during sepsis[39, 40].In our study, we found BTLA-expressing mDCs from septic neonates indeed secreted lower levels of TNF-α and IL-12, but higher IL-10 after infected by E.coli. However, study with acute experimental sepsis induction in mice model demonstrated that IL-10, TNF-α, IFN-γ and IL-6 all were increased, whereas only IL-10 expression was reduced when diminished BTLA expression. In other words, BTLA expression only contributes to the regulation of anti-inflammatory system but not pro-inflammatory system. There are potentially many reasons for why the different results occurred. Firstly, the sample collection time in mice could be chose, like that study was in 24 h post-CLP; however, it was not possible to establish the exact time-point that the patient’s septic insult and pathogen challenge began. Also, the nature and source of the initial nidus of infection in those neonates included into our study were differed. Therefore, BTLA still could be an important regulatory protein on mDCs. However, further studies should be proceeded to investigate the potential molecular mechanism of how BTLA regulate the mDCs function to regulate the sepsis outcome in neonates.