Background
Papillon-Lefèvre syndrome (PLS) is a hereditary rare autosomal recessive condition affecting up to 5 people per million. An ectodermal dysplasia caused by loss of function mutations of the cathepsin C gene (
CTSC), PLS is manifested as periodontitis and skin lesions [
1]. It is remarkable that the PLS-associated and rapidly progressing severe periodontitis affects not only the primary dentition, but extends to the permanent dentition as well, leading to premature loss of both primary and permanent teeth. The other manifestation of PLS syndrome is a diffuse palmoplantar keratoderma characterized by hyperkeratosis and erythema on the palms, soles of the feet, elbows and knees [
2‐
5]. Generally, the cutaneous lesions of PLS first appear between 6 months and 4 years of age, but in rare cases they may emerge in the first 3 months of life. Moreover, several cases of late-onset PLS have also been reported in the literature [
6‐
8].
The etiology of PLS is not completely understood [
9]. The primary cause of PLS has been identified as mutations in the
CTSC gene, in the human chromosomal region 11q14.1-q14.3. CTSC is a lysosomal cysteine protease, also known as dipeptidyl peptidase 1 (DPPI). More than 100 mutations have been described including missense, nonsense or frameshift variants, which result in a reduced CTSC activity and coupled reduced host response against bacteria [
10‐
14].
The
CTSC gene is highly expressed in many tissues, such as the epithelial regions of the palms, soles and knees; keratinized oral gingival fibroblasts and osteoclasts [
10,
15‐
17]. The majority of proteins that require processing by CTSC are part of the innate immune system [
18]. Affected immune cells include natural killer cells, polymorphonuclear cells and T lymphocytes [
19]. This may explain the multidimensional symptoms of PLS and may contribute to the development of malignant cutaneous neoplasms in certain PLS cases [
20].
Polymorphonuclear neutrophils (PMNs) play a major role in the immune response against the key pathogens of various forms of periodontitis. Three strategies by which neutrophils serve as a first line of defence against invading pathogens are known as follows: i.) secretion of antimicrobial peptides (degranulation); ii.) engulfment of bacteria (phagocytosis); iii.) release of neutrophil extracellular traps (NETs) consisting of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) capable of capturing and killing pathogenic microorganisms [
21].
Human neutrophils have an ambivalent role in the interactions with
Aggregatibacter Actinomicetemcomitans (Aa). Apparently, Aa. exploits neutrophils by inducing the exocytosis of azurophilic granules and the release of epinephrine, a catecholamine which activates QseBC bacterial two-component signalling system, facilitating thereby the survival and growth of
A. actinomicetemcomitans under anaerobic conditions [
22].
A. actinomicetemcomitans produces stabile biofilms that are resistant against removal by the flow of the saliva or the gingival crevicular fluid and highly resistant to antimicrobials as well, partly due the expression of the histone-like (H-NS) family of nucleoid-structuring proteins which facilitate the formation of multispecies biofilms [
23]. In contrast LL-37, a peptid processed by protease cleavage of human CAP-18 (cathelicidine antimicrobial peptid 18) suppresses biofilm in vitro biofilm formation. LL-37 antimicrobal protein released from azurophilic granules of PMNs, is capable to bind and disrupt the membrane of various
A. actinomycetemcomitans strains
. Furthermore, LL-37 in sublethal dose also markedly suppressed in vitro biofilm formation and demonstrates opsonisation and agglutination enhancing thereby bacterial clearance by neutrophils and macrophages [
24]. Thus evidence shows that LL-37 may act not only as a direct antimicrobial peptide, but also by its antibiofilm and immuno-modulatory properties.
CTSC is essential for the activation of three serine proteases (proteinase 3, neutrophil elastase and cathepsin G), which are components of the azurophilic granules of PMNs. Neutrophil azurophilic granules are partially responsible for the intracellular destruction of phagocytosed pathogens. Neutrophils are also a rich source of antimicrobial peptides (α-defensin 1-4 and cathelicidin). The human cathelicidin (hCAP18/LL37) gene (
CAMP, cathelicidin antimicrobial peptide) encodes the peptide precursor human cationic antimicrobial protein (CAP), which is stored in the secondary granules of neutrophils as an inactive pro-form and gains antimicrobial activity via proteinase-3 cleavage, which generates peptide LL-37 - C-terminal part of the only human cathelicidin. Granular enzymes and peptides including neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, leukocyte proteinase 3 (PR3), lactoferrin, gelatinase, lysozyme C, calprotectin, neutrophil defensins and cathelicidins are also involved in the antimicrobial activity of NETs [
21].
The lack of functional CTSC in PLS patients leads to the absence of LL-37 in the gingival region [
16,
25,
26]. PMNs are the main source of LL-37 in the healthy periodontium [
26]. The levels of antimicrobial peptides in gingival crevicular fluids correlate with the prevalence and quantity of oral pathogens, predominantly
A. actinomycetemcomitans in the subgingival plaque of PLS patients [
24‐
26]. It is also noteworthy that bacterial proteases of other important periodontopathogens, such as
Porphyromonas gingivalis,
Tannerella forsythia, and
Treponema. denticola might degrade hCAP18/LL-37 [
26]. Severe congenital neutropenia (SCN, morbus Kostmann) is an autosomal recessive hereditary disease characterized by severe neutropenia resulting in LL-37 deficiency. Lack of LL-37 or very low levels of LL-37 in plasma, saliva and neutrophils of patients with Kostmann disease is associated with oral infections/inflammatory diseases like chronic periodontal disease. Although these patients are successfully treated by recombinant granulocyte-colony stimulating factor, which restores their levels of neutrophils, they have extremely low levels of both LL-37 and its precursor hCAP18 in plasma, saliva and neutrophils. For this reason, they present with symptoms of recurrent infections and periodontal disease [
27]. A mutation causing a disruption of the neutrophil elastase enzyme was identified in the previously mentioned SCN patients. In another group of patients with Kostmann syndrome, we find a homozygous mutation of the
HAX1 gene that regulates neutrophil apoptosis, which is the most common SCN-related mutation in Turkey, and there is a variant in which neutropenia is not associated with the symptoms of Kostmann syndrome [
28,
29]. Despite the fact that the level of antimicrobial proteins (HNP1-3, LL-37) in patients treated with G-CSF did not differ compared to the control group, these patients show more severe inflammatory signs, low bacterial diversity with high bacterial load associated with dysbiosis [
29]. Severe congenital neutropenia treated by bone-marrow transplant may result, however, in functionally intact neutrophils and normal levels of LL-37 in plasma, saliva and neutrophils and, furthermore, periodontally healthy patients [
30‐
32].
The evidence shows that LL-37 deficiency, either due to a deficiency of the enzyme required for the formation of the active form (PLS syndrome), or due to a low number or damaged neutrophils (Kostmann disease), causes severe, rapidly progressive, chronic periodontitis.
Numerous microbiological studies have identified the composition of the subgingival biofilms but there is limited information about the microbiological profiles of periodontal lesions in PLS. Species diversity was analysed with various microbiological approaches. Clonal analysis already indicated the importance of
A. actinomycetemcomitans in the pathogenesis of PLS [
25], which has been supported by increased levels of specific antibody against
A. actinomycetemcomitans [
2,
33]. Classical microbiological studies corroborated the outstanding predominance of
A. actinomycetemcomitans in the subgingival plaques of PLS patients [
26,
34‐
37]. Serine proteases, such as CTSC, are keystone enzymes in controlling the
A. actinomycetemcomitans invasion. Low levels of LL-37 will render PMNs incapable of effectively neutralizing the leukotoxin produced by
A. actinomycetemcomitans, further facilitating the aggression of
A. actinomycetemcomitans in the subgingival plaque [
16,
25,
38,
39]. Leukotoxin produced produced by
A. actinomicetemcomitans causes activation of caspase-1 via NLRP-3 (NLR Family Pyrin Domain Containing 3) inflammasome which converts pro-IL-1β into active IL-1β and mediates its release from human macrophages, resulting in osteoclast differentiation and bone resorption. This mechanism correlated the onset and progression of periodontitis [
40,
41].
Periodontitis is an inflammatory disease caused primarily by periodontopathogenic microbiota, whereas host defence mechanisms play an important role in its pathogenesis by modulating the local infection and eliciting the breakdown of periodontal tissues [
42,
43].
Intrafamilial transmission of periodontopathogenic microorganisms is a well-known phenomenon [
44‐
46]. The family involved in our investigation, was composed of both PLS patients carrying a seven-bases deletion in the
CTSC gene and periodontally healthy members [
47]. In this study we aimed the comprehensive characterization of the subgingival plaque microbiota of this family. We present the data on 3 siblings and 2 parents: two of the sisters developed typical symptoms of PLS, while the oldest sister and the parents were phenotypically healthy [
47]. We compared the microbiota in samples from the PLS family with oral microbiota of adult chronic periodontitis patients as well as with data on samples from adolescents with and without gingivitis, presented earlier [
48,
49]. Finally, we have also evaluated the clinical and microbial results of non-surgical periodontal treatment and adjunct antimicrobial therapy, applied in the case of the two sisters with PLS, designated as Patient-A and Patient-B respectively.
Discussion
It was suggested that immune defects affecting neutrophils play an important role in the pathology of periodontal disease [
67]. Our study indicates that – possibly on the basis of, or in addition to such dysregulated immune alterations - in PLS patients one may observe an altered composition and a decline in the diversity of the subgingival microflora compared to healthy family members. In PLS patients we detected a higher relative abundance of several periodontal pathogens, especially
A. actinomycetemcomitans, which increased after mechanical and antimicrobial therapy. Our results demonstrated that antibiotics could enhance the beneficial clinical effects of mechanical periodontal therapy, and improve the clinical outcome, only in patients with good oral hygiene compliance. We observed that although the healthy subjects had quite good clinical parameters, composition of subgingival microflora consisted predominantly of periodontal pathogens. Thus, they fell within the “moderately diseased periodontitis” cluster.
The oral phenotype of the two PLS sisters in this study partly followed the syndrome profile but also showed considerable differences between the affected individuals. Although the oral habits changed in case of the older affected (Patient-B), the relatively healthy periodontal conditions were maintained for a prolonged period thanks to the premature loss of deciduous teeth and a consequent “microbial shift”, in accordance with the conclusions of several studies [
68‐
71]. Early loss of primary teeth influences the development of jaw, the position of the lip as well as the position of the permanent teeth underlying the need for orthodontic treatment [
72].
In our case, the early regular periodontal treatment of younger affected sister (Patient-A) resulted in the maintenance of deciduous teeth prior to the eruption of permanent teeth. Deep periodontal spaces developed during eruption at the adjacent primary teeth after exfoliation of secondary teeth, providing opportunity to infection. Despite of meticulous individual and professional oral hygiene and periodontal treatment, the periodontal destruction of the younger affected patient (Patient-A) became more severe relative to her sister (Patient-B).
In previous studies, various methods were applied to analyse distinct microbiomes in PLS patients. Albandar et al. presented a subgingival composition of microflora in PLS patients, based on clonal and microarray analysis of 16S rRNA genes [
34], whereas Lettieri described the salivary microbiome of PLS patients by next generation sequencing [
33]. In line with these early studies, we also noted a higher relative abundance of
A. actinomycetemcomitans, T. forsythia, F. nucleatum, P. intermedia in the PLS samples [
33‐
37]. We also found, however, a higher proportion of
P. oris,
P. micra and,
C. Saccharibacteria. Furthermore, the relative abundance of
A. actinomycetemcomitans was significantly higher in the PLS patients in our study relative to previous reports [
2,
6,
36,
37]. From the above studies one can conclude that
A. actinomycetemcomitans is the chief oral pathogen responsible for the periodontal disorders of the PLS patients.
As far as we know, a complete microbiological profile of healthy PLS family members has not been presented yet. We compared the oral microbial profile of healthy and diseased PLS family members with those of periodontitis patients as well as adolescents with gingivitis and gingival health, reported earlier [
48,
49]. Our results is consistent with the findings of previous investigations, which reported the presence of pathogenic bacterial species in periodontally healthy individuals [
73,
74]. Stabholz et al. reported high prevalence of leukotoxic strains of
A. actinomycetemcomitans in unaffected members of PLS family. Our results are not consistent with these findings, the occurrence of
A. actinomycetemcomitans was relatively low in healthy family members, but the other important periodontopathogens isolated in higher proportions of affected samples (
T. forsythia, F. nucleatum, P. intermedia, P. oris,
P. micra and,
C. Saccharibacteria) are also notable components of microbiome of healthy family members. This may be due to familial aggregation of periodontal pathogenes, but it seems that
A. actinomycetemcomitans may be an intrinsic feature of this disease rather than solely inherited from parents, as previously published some authors [
44‐
46,
75].
Several PLS patients have been reported to lack notable immunodeficiency in spite of the missing functional serine protease in PMNs and cytotoxic lymphocytes [
76,
77]. The reduced capacity of NET production and higher reactive oxygen species (ROS) formation provide stimulus for the improper recruiting of highly responsive neutrophils in periodontal tissues, acute inflammation and bone loss [
78]. Acute inflammatory episodes and recurrent pyogenic infections may occur in PLS patients [
50] as we experienced at Patient-B.
Non-surgical periodontal treatment with adjunctive systemic medication is an important therapeutic approach for treatment of active periodontal inflammation [
79,
80]. Compared to her elder sister (Patient-B) the periodontal parameters of the younger affected patient (Patient-A) were substantially better as the result of the combined periodontal treatment. Before periodontal therapy Patient-B harboured a less diverse subgingival microbiome. Following the mechanical debridement and low-dose tetracyclin treatment, the composition of subgingival microflora became less diverse, the relative abundance of
A. actinomycetemcomitans elevated to 62%,
P. oris increased while
P. intermedia decreased. Our findings demonstrated a short-term effect of mechanical and antimicrobial therapy. A possible explanation for these apparently unfavourable results could be ineffective oral hygiene, as Patient-B reportedly lost her motivation in personal oral hygiene [
80]. Additionally, the systemic antibiotic treatment could induce the emergence of pathogens resistant to antibiotics. Kleinfelder et al. have reported the increased proportion of
A. actinomycetemcomitans in the microflora of a PLS patient no later than 8 weeks after administration of metronidazole. The repeated medication may result in continuous emergence of resistant microorganisms in the subgingival microbiomes [
6]. Jepsen et al. studied the antimicrobial susceptibility of selected periodontopathogens. It is noteworthy, however, that
A. actinomycetemcomitans is not susceptible to metronidazole [
81]. Although
A. actinomycetemcomitans is susceptible to Doxycyclin the increased relative abundance of
A. actinomycetemcomitans after the adjunctive administration of this active agent combined with mechanical treatment may suggest that medication with systemic antibiotics at patient with poor oral hygiene result in failed clinical and microbial values, furthermore, likely facilitate selecting of resistant bacterial strains. At any rate, the teeth of Patient-B were saved with moderate attachment loss at the age of 18 years.
A generalized conclusion of this study suggests that the PLS patients apparently differ substantially from subjects of common periodontal inflammatory diseases. An equally important finding indicated that the clinically healthy recessive heterozygote carriers of the CTSC gene mutations show the alterations in the microbiome composition characteristic of mild periodontitis. These observations may trigger the development of specific treatment strategies, e.g., specific probiotic strains/bacteriocins against
A. actinomycetemcomitans. and to develop more efficient therapeutic protocols for the affected patients. In recent years there have been number of antimicrobial peptids entering clinical trials to treating wound healing in patients with immunodeficiency. The peptide omiganan (MBI226), a12-residue amide derivative of indolicidin (a cathelicidin isolated from bovine neutrophils), and several peptide mimetics are the potential active agent for topical treatment of skin lesion. It is advisable to consider the use of these active substances in controlled-release local delivery antimicrobials in periodontal pockets [
82,
83]. Until then, the frequent, meticulous regular mechanical periodontal treatment with deliberated adjunctive systemic administration of antibiotics combined with good individual oral hygiene established with motivation and instruction is an essential condition for successful maintenance therapy. Continuous periodontal maintenance therapy of healthy family members should be considered to avoid cross-infections.
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