Discussion
In
order to avoid the influence of glucocorticoid and any other
immunosuppressant on the main outcome measures in this study, all the 40
selected patients with active MPO-AAV were initially diagnosed without
any treatment with these agents. Among them, 39 patients (97.5%) had
MPA, which was consistent with the fact that MPA with MPO-ANCA was most
common AAV in China and other East Asian countries[4]. Also, Age and
gender distribution in our study was respectively comparable between the
patient and HC group as statistical analysis showed there was no
significant difference for them between the 2 groups (Table I).
PAD4 is one of the 5 isoenzymes of PAD (PAD1-4 and PAD6) in human, a
family of Ca2+-dependent deiminase enzymes. It is
mainly expressed in the nucleus of neutrophil [27, 28]. Li et al.
constructed PAD4 knockout mice and stimulated them with chemokines and
bacteria. They found chromatin decondensation was inhibited in
neutrophils, NETs could not form and the antimicrobial ability of mice
was significantly decreased, which indicated PAD4 played an important
role in formation of NETs by mediating histone citrullination[29].
Studies shown that PAD4 was also involved in the pathogenesis of both
tumor and autoimmune diseases, such as SLE and rheumatoid arthritis (RA)
[21, 30-32]. Knight et al. published that MRL/lpr SLE model mice
treated by PAD4 inhibitors presented lighter clinical manifestations and
disease activity compared to the controls[21]. Moreover, PAD4 in
synovial fluid of RA was detected at significantly higher levels than
that of osteoarthritis or psoriatic arthritis, which might be account
for the generation of autoantigens in RA[33]. In this study, we
detected the expression level of PAD4 in neutrophils by FCM, and found
both the percentage of PAD4+ neutrophils and the MFI
of PAD4+ neutrophils were significantly higher in
patient group than in HC group, respectively (Fig.1). This might account
for the NETs in patient group was markedly higher than that in HC group
(Fig. 2A).
Further
mover, the percentage of PAD4+ neutrophils and the MFI
of PAD4+ neutrophils respectively manifested positive
correlation with NETs in patient group (Fig.3A and 3B).
As for NETs, in addition to its basic function of innate immunity
against microbial infection[14], it was found to play important
roles in pathogenesis of various autoimmune diseases, especially of AAV,
by promoting both innate and adaptive immunity pathway. In a vitro
experiment, Wang et al. demonstrated NETs could activate the cAP, an
innate immune pathway to aggravate inflammation by producing C5a
[18]. In AAV, the most common autoantigens (MPO and PR3) were the
component of NETs[14], Sangaletti et al. reported that autoantigens
in NETs could be processed and present to adaptive immune T cell more
effectively by myeloid dendritic cells to induce the production of
autoantibodies including MPO-ANCA[34]. MPO-ANCA combined with MPO on
neutrophils and promote the neutrophils to discharge granules, to
produce reactive oxygen radicals (ROS), to form NETs and finally to
damage the tissues and organs by the inflammatorily destroyed
vessels[15, 16]. The discharged granules from the neutrophils
provoked by MPO-ANCA contained factors, such as properdin, that could
boost cAP to present more C5a[35, 36]. In this study, we found serum
C5a of patient group was significantly higher than that in HC group,
which supported the activated cAP in active MPO-AAV (Fig. 2B). Although
Wang et al. reported NETs could activate the cAP[18], our study
indicated that, compare to NETs, MPO-ANCA might play more important
roles to activate cAP by releasing granules because C5a was
significantly associated with MPO-ANCA but not with NETs (Fig. 3C and
Fig. 3D). Our result also showed MPO-ANCA had significant positive
correlation with NETs (Fig.3E), which might be attributed to that MPO in
NETs could be more effectively processed and present as autoantigen by
myeloid dendritic cells to activate adaptive immunity[34]. Kusunoki
et al. found in their mice model of MPO-AAV that histone citrullination
could decrease by inhibiting PAD4 with Cl-amidine, and then serious
MPO-ANCA could also be reduced [37].
As discussed above, PAD4 played a key role in NETs formation, which
might promote adaptive immunity to facilitate the generation of MPO-ANCA
and also might activate cAP to increase C5a. Both MPO-ANCA and C5a might
trigger neutrophils to form NETs, to accelerate cAP, to produce ROS and
etc. This then could create a self-fueling inflammatory amplification
loop, a “vicious cycle”, to aggravate clinical inflammatory damage of
MPO-AAV. Actually, Univariable correlation analysis in this study showed
that the percentage of PAD4+neutrophils, MFI of
PAD4+neutrophils, NETs, C5a and MPO-ANCA was
positively correlated with BVAS, respectively (Tab. II). However,
multivariable correlation analysis revealed that only the percentage of
PAD4+neutrophils and MPO-ANCA was independently
correlated with BVAS, respectively (Tab. II). In fact, MPO-ANCA was the
hallmark for MPO-AAV as an autoimmune disease. Previous studies had
demonstrated MPO-ANCA as a pathogenic autoantibody by experimental
research and clinical report[8, 38]. Though PAD4, NETs, C5a and the
“vicious cycle” among them might contribute to the production of
MPO-ANCA, MPO-ANCA present as an independent factor affecting the
vasculitis activity in this clinical study. Meanwhile, PAD4 could have
independent influence on BVAS, which should be attributed to its
enzymatic function in NETs generation and the NETs pathologic efficacy
in MPO-AAV.