DISCUSSION
Past clinical experience has shown that patients with sepsis benefit
from de-escalation antibiotic therapy, but the mechanism has not been
explored in depth. This study is the first time to elaborate the
mechanism of DE therapy from animal experiments. Our results show that
different β-Lactams have different effects on NETs formation bothex vivo and in vivo , and we demonstrated that
de-escalation antibiotic therapy, including the early use of
NET-promoting antibiotics and the late use of NET-inhibiting
antibiotics, could fully exploit the immunomodulatory effects of NET
formation during the different stages of sepsis. More importantly, these
results underline the important role of NETs directed initial empirical
antibiotic therapy in the infection of polymicrobial sepsis in
vivo .
Similar with previous studies, our data emphasize the critical
antibacterial role of NETs in the early stage and the deleterious role
of NETs in the late stage of sepsis [17, 19]. Neutrophils serve as
the first line of defense in the innate immune system against bacterial
pathogens. The release of intracellular contents, which is called
neutrophil extracellular traps, is critical for killing bacteria in
infection and sepsis [3]. Current studies have shown that NETs and
their elements can cause organ injury, coagulation disorders, autoimmune
diseases, and thromboembolism [20]. Although NETs are a double-edged
sword in sepsis, few studies have focused on their antibacterial
function in the early stage of sepsis, during which NETs can trap
pathogens and prevent bacterial dissemination [17, 18, 21]. Our data
show clearing NETs with DNaseI will lead to the spread of infection and
increased organ damage, these results prove that NETs formation playing
an important role in organ protection during the early stage of sepsis.
We demonstrated, for the first time, that in addition to macrolide and
quinolone drugs, β-lactam antibiotics can also modulate the formation of
NETs in vivo and in vitro . Previous studies have proven
that some antibiotics may exert immunomodulatory functions, and with the
discovery of NETs, more antibiotics have been found to affect the immune
system [22-24]. However, no studies have shown immunomodulatory
functions of β-lactam antibiotics, and some studies have shown a lack of
immunomodulatory function of β-lactam antibiotics. This study has proven
that different subgroups of β-lactam antibiotics have distinct effects
on NET formation and that imipenem and ceftriaxone regulate NETs in a
ROS-dependent manner. ROS generation is a well-recognized step in NET
formation, and many studies have shown that NET-associated diseases may
be alleviated by the usage of ROS scavengers or inhibitors [25]. The
β-lactam antibiotic-induced NET regulation observed in this study could
be inhibited by NOX inhibitors or promoted by exogenous ROS, which
confirmed the involvement of ROS.
De-escalation therapy has been shown to be associated with reduced
mortality in patients with severe sepsis and septic shock and has been
shown to be effective and safe; however, there is no adequate, direct
evidence or high-quality RCT studies to prove these claims, and the
necessary studies to elucidate the mechanism of de-escalation therapy
have not been conducted [26]. Although there is no consensus when
the ridge line of early and late stage of sepsis is, it is acknowledged
that the pathogen confirmed in 24-72h after sepsis diagnose. Thus, in
our study we defined that 3 days were the early stage in according to
the unknown pathogen period and discover whether the initial empiric
antibiotic act differently. We confirmed that although it cannot improve
the 6-day mortality of CLP mice in our study, de-escalation therapy can
reduce organ injury and inflammatory responses, which is consistent with
observations in human patients. According to the guidelines of sepsis
treatment, antibiotics were administered 1 h after the CLP procedure and
de-escalated. Escalation therapy was used under the guidance of
empirical antibiotic usage in our study. We showed better reductions in
the AST, ALT, serum creatine, and cytokine levels (IL-6, IL-10, TNF-α,
IFN-γ, MIP-2 and MCP-1) in both the early and late stages of sepsis.
Based on the recovery of the function of β-lactam antibiotics in the
regulation of NETs, we then explored the mechanism of de-escalation
therapy. In our study, we found that the combination of different
antibiotics affects the serum levels of MPO-DNA, which is a biomarker of
NETs. With the sequential treatment with NET-promoting and
NET-inhibiting antibiotics, NET formation could be regulated, ultimately
maximizing the function of NETs and reducing their damage. In the
experiment with imipenem-ceftriaxone de-escalation therapy, NET
formation was higher in the early stage and lower in the late stage,
which was accompanied by milder organ injury and inflammatory responses.
In addition, the administration of DNase I with de-escalation therapy
reduced the formation of NETs, but organ injury and inflammatory
responses were exacerbated. To confirm the role of NETs, we conducted
histological and immunohistochemical staining, and with greater
infiltration of citH3, which is a credible NET biomarker, increased
injury to organs and enhanced apoptosis of tissue cells were observed.
It seems, therefore, that in addition to the useful antibacterial
function of antibiotics, the function of de-escalation therapy in the
regulation of NETs is more important in sepsis; this regulatory role
enhances the antibacterial function of innate immunity and prevents
further NET-associated damage in sepsis. Although there was no
significant difference in survival between the DE group and the ES group
due to sample size. Meanwhile, several therapeutic strategies such as
anti-infection, nutrition and organ function support are employed in
clinical treatment of sepsis, and antibiotic treatment alone does not
reflect the overall picture of sepsis treatment. However, organ damage
and the levels of inflammatory cytokines in the DE group were
significantly decreased compared to the ES group. These results show the
benefits of de-escalation antibiotic therapy strategies in sepsis.
However, there are some limitations. Polymicrobial sepsis model was used
and we did not examine the bacterial loads and differentiate bacterial
infiltration. It is important to detect the antibacterial spectrum,
which takes about 24-72h to confirm in clinical practice. The initial
administration of different antibiotics may shift bacterial colonization
with a certain resistance to NET-dependent killing and influence the
real outcome. Besides, bacterial colonization changes may also affect
homeostasis in intestine which contribute to the injury of intestine.
Those speculative assumption need to be addressed in future studies.
Lastly, we assessed the 6-day mortality in the DE group which was
shorter compared with the real clinical setting. Extended observation
time may be expected.