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Modulation of inflammasome activity by Porphyromonas gingivalis in periodontitis and associated systemic diseases

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Inflammasomes are large multiprotein complexes localized in the cytoplasm of the cell. They are responsible for the maturation of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 as well as for the activation of inflammatory cell death, the so-called pyroptosis. Inflammasomes assemble in response to cellular infection, cellular stress, or tissue damage; promote inflammatory responses and are of great importance in regulating the innate immune system in chronic inflammatory diseases such as periodontitis and several chronic systemic diseases. In addition to sensing cellular integrity, inflammasomes are involved in the homeostatic mutualism between the indigenous microbiota and the host. There are several types of inflammasomes of which NLRP3 is best characterized in microbial pathogenesis. Many opportunistic bacteria try to evade the innate immune system in order to survive in the host cells. One of these is the periodontopathogen Porphyromonas gingivalis which has been shown to have several mechanisms of modulating innate immunity by limiting the activation of the NLRP3 inflammasome. Among them, ATP-/P2X7- signaling is recently associated not only with periodontitis but also with development of several systemic diseases. The present paper reviews multiple mechanisms through which P. gingivalis can modify innate immunity by affecting inflammasome activity.
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REVIEW ARTICLE
Modulation of inflammasome activity by Porphyromonas
gingivalis in periodontitis and associated systemic
diseases
Ingar Olsen
1
* and O
¨zlem Yilmaz
2
1
Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway;
2
Department of
Oral Health Sciences, Medical University of South Carolina, Charleston, SC, USA
Inflammasomes are large multiprotein complexes localized in the cytoplasm of the cell. They are responsible for
the maturation of pro-inflammatory cytokines such as interleukin-1b(IL-1b) and IL-18 as well as for the
activation of inflammatory cell death, the so-called pyroptosis. Inflammasomes assemble in response to cellular
infection, cellular stress, or tissue damage; promote inflammatory responses and are of great importance in
regulating the innate immune system in chronic inflammatory diseases such as periodontitis and several chronic
systemic diseases. In addition to sensing cellular integrity, inflammasomes are involved in the homeostatic
mutualism between the indigenous microbiota and the host. There are several types of inflammasomes of which
NLRP3 is best characterized in microbial pathogenesis. Many opportunistic bacteria try to evade the innate
immune system in order to survive in the host cells. One of these is the periodontopathogen Porphyromonas
gingivalis which has been shown to have several mechanisms of modulating innate immunity by limiting the
activation of the NLRP3 inflammasome. Among them, ATP-/P2X
7
- signaling is recently associated not only
with periodontitis but also with development of several systemic diseases. The present paper reviews multiple
mechanisms through which P. gingivalis can modify innate immunity by affecting inflammasome activity.
Keywords: innate immunity;subversion;inflammasomes;inflammation; Porphyromonas gingivalis; persistence;periodontitis;
systemic diseases
Responsible Editor: Daniel Smith, The Forsyth Institute, Boston, United States.
*Correspondence to: Ingar Olsen, Department of Oral Biology, Faculty of Dentistry, University of Oslo, P. B.
1052 Blindern, NO-0316 Oslo, Norway, Email: ingar.olsen@odont.uio.no
Received: 12 November 2015; Revised: 6 January 2016; Accepted: 6 January 2016; Published: 4 February 2016
The innate immune system is the first line of de-
fense against microbial pathogens. It is initiated
by genome-encoded pattern recognition receptors
(PRRs) that respond to invading microorganisms. PRRs
recognize microbial pathogen-associated molecular pat-
terns (PAMPs). This leads to activation of host defense
pathways to clear the infection (1). In addition to micro-
bial components, the receptors can respond to danger-
associated molecular patterns (DAMPs) derived from
the host (ATP, DNA, cholesterol crystals). Also envi-
ronmental irritants such as asbestos, silica, alum, and
nanoparticles can stimulate inflammasome-driven inflam-
mation (2, 3). In the detection of pathogens, toll-like
receptors (TLRs) are of crucial importance. They recog-
nize distinct PAMPs and participate in the first line of
defense against invading pathogens, playing a signifi-
cant role in inflammation and immune cell regulation.
Other well characterized evolutionary conserved PRRs
involved in innate immune defense are retinoic acid-
inducible gene-I (RIG-I) receptors, C-type lectin recep-
tors (CLRs), and nucleotide-binding domain (NOD)-like
receptors (4, 5). All these receptors are expressed by
several cell types such as macrophages, neutrophils,
monocytes, and epithelial cells (6).
The activation of PRRs and their post-receptor sig-
naling can stimulate recruitment of the so-called in-
flammasome complexes (1, 3, 615). Inflammasome is
a relatively new concept introduced by Tschopp et al. in
2002 (7). Later, Tschopp also introduced the concept
of metabolic syndrome that senses metabolic stress and
contributes to the metabolic syndrome associated with
obesity and type 2 diabetes (16).
The aims of the present paper are to give a brief
description of inflammasome compositions and functions
and to systematically review how Porphyromonas gingivalis,
a putative keystone pathogen in chronic periodontitis (17),
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Journal of Oral Microbiology 2016. #2016 Ingar Olsen and O
¨zlem Yilmaz. This is an Open Accessarticle distributedunder the terms of the Creative Commons
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reproduction in any medium, provided the original work is properly cited.
1
Citation: Journal of Oral Microbiology 2016, 8: 30385 - http://dx.doi.org/10.3402/jom.v8.30385
(page number not for citation purpose)
well known for its way to manipulate the innate immune
system (18), may carry out/modulate inflammasome
activation in chronic periodontitis and certain other
chronic systemic infections. The role of inflammasome
and danger molecule signaling in the oral cavity was
recently reviewed by Yilmaz and Lee (6).
Inflammasomes
Inflammasomes are multiprotein complexes localized
within the cytoplasm of the cell. They are engaged in
the maturation of pro-inflammatory cytokines such as
interleukin-1b(IL1-b) and interleukin-18 (IL-18) (19).
After infection or cellular stress, inflammasomes are
assembled, activated, and involved in host defense and
in the pathophysiology of diseases (20). Inflammasomes
follow canonical or non-canonical pathways. A typical
functional canonical inflammasome complex consists of
a nucleotide-binding leucine-rich repeat (NLR) protein,
an adaptor molecule apoptosis-associated speck-like
protein containing a caspase activation and recruitment
domain (ASC) and procaspase-1 (21). A typical non-
canonical inflammasome is the one activating caspase-11,
which so far is an understudied pro-inflammatory
caspase (22).
A unique scaffolding protein (NLR) dictates the for-
mation of inflammasomes. Mutations in members of the
NLR family have been linked to various inflammatory
diseases consistent with the fact that these molecules play
an important role in hostpathogen interactions and the
inflammatory response (23). Each inflammasome con-
tains a unique sensor protein of the NLR superfamily or
the PYRIN and HIN-200 domain-containing (PYHIN)
superfamily (10). In NLRs, signaling is exerted by cas-
pase activation and the so-called caspase activation and
recruitment domains (CARDs). These can recruit caspase-1
directly or by PYRIN domains recruiting caspase-1 via
the CARD-PYRIN-containing adaptor protein ASC (10).
The adaptor protein mediates a critical step in innate
immune signaling by bridging the interaction between
the pathogen recognition receptors and caspase-1 in
inflammasome complexes (24).
Inflammasomes have a critical role in initiating innate
immune responses, particularly by acting as platforms for
activation of the inflammatory caspase proteases. Among
these, caspase-1 initiates innate immune responses by
specifically cleaving of pro-IL-1band pro-IL-18 and
mediates their maturations and release (10). These cyto-
kines promote recruitment of phagocytes, angiogenesis,
epithelial cell repair, and regulation of cytokines and
chemokine production by other immune cells at the site
of infection or injury (reviewed by Hao et al. (25)).
Inflammasomes also take part in the host defense
independent of their classic cytokine targets IL-1band
IL-18.
Caspase-1 and 11 can start a rapid and inflammatory
form of cell death, the so-called pyroptosis. They are
distinct from caspases classically involved in apoptosis.
Pyroptosis, a program of cellular self-destruction that is
intrinsically inflammatory, results from osmotic pressure
created by caspase-1-dependent formation of membrane
pores (26, 27) and is associated with rapid release
of cytosolic contents. This process can restrict intracel-
lular replication of invasive bacterial pathogens (28) and
probably acts in synergy with the recruitment of neu-
trophils by IL-1bto restrict replication of bacteria
in vivo (10).
Inflammasomes sense cellular integrity and tissue
health. When cell homeostasis is disrupted, inflammation
is caused by the release of cytokines. A large amount of
infectious and noxious insults can assemble these special
structures. Thus inflammasomes may have a role in
bacterial, parasitic, fungal, and viral infections (29).
Inflammasomes also sense products and endogenous
signals that indicate loss of cellular homeostasis (10, 15)
and can be active both in periodontitis and several
systemic diseases (6, 30).
Several distinct inflammasomes have currently been
described, each of which is activated by unique stimuli
(Fig. 1). Inflammasomes such as NLRP1 (nucleotide-
binding domain-like receptor protein) inflammasome1,
NLRP3, NLRP4, NLRP6, NLRP/NLRP12, and AIM2
(Absent In Melanoma 2) have been recognized acting in
the host defense against intracellularly invading patho-
gens. Some inflammasomes are particularly well char-
acterized for their role in bacterial recognition such as
NLRC4, NLRP3, and AIM2 (Fig. 1). Also there may be
more paths to IL-1band IL-18 generation than via
caspase-1 (1). It is likely that other caspase-1-dependent
effector cytokines are produced by other proteases during
infection (31). Several pathogens have been found to
develop strategies to counteract inflammasomes (the
so-called pathogenic stealth mechanisms) (32). Thus,
Staphylococcus aureus can modify its cell wall peptido-
glycan to prevent degradation by lysozymes through
peptidoglycan O-acetyl transferase A which also strongly
suppresses inflammasome activation and inflammation
in vitro and in vivo (33).
While inflammasome activation is important to host
defense, excessive inflammasome activation can be detri-
mental to health. Inflammasome hyperactivation is re-
cently proposed to be the basis for autoinflammatory
disease pathogenesis, whereas inflammasome regulated
activity is central for appropriate host defense and protec-
tion from sepsis (16). Accordingly, there is need for a
balance between resolution of infection and excessive
inflammation (1). It should also be kept in mind that
several pathogenic bacteria, for example, Yersinia pestis,
Salmonella, and Listeria monocytogenes activate mul-
tiple inflammasomes demonstrating redundancy of
Ingar Olsen and O
¨zlem Yilmaz
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inflammatory receptors in vitro and in vivo (34, 35). It is
also possible that several NLRs, AIM2s, and caspases
co-operate during infections (1), which may be necessary
for optimal responses to be obtained.
In inflammasome-mediated cytokine release, a multi-
step activation pathway is followed. First, there is an NF-
kB-dependent upregulation of the inactive pro-forms
of IL-1band IL-18 and of some NLRs like NLRP3;
thereafter, activation of the NLR or AIM2, and inflam-
masome formation occurs (1). Some cells have simpler
activation pathways because they have higher basal levels
of the pro-forms of IL-1band IL-18.
Interestingly, inflammasomes are also involved in the
homeostatic mutualism between host and commensals. In
the intestine, one inflammasome function is to control the
composition of the microbiota (36). The NLCP4 inflam-
masome, expressed by intestinal phagocytes in particular,
plays a major role to discriminate between commensal
and pathogenic microbes and initiate a harmful res-
ponse to the latter (37). Thus, inflammasomes may have a
variety of roles regulating homeostasis in the intestinal
tract and microbial ecology preventing the emergence of
pathobionts (25, 38). Inflammasome-deficient mice ex-
hibited an aberrant microbial community that triggered
an enhanced inflammatory reaction in the intestine (39).
This microbial dysbiosis affected the physiology and
pathophysiology both locally in the intestine and sys-
temically and might contribute to the pathogenesis of
intestinal bowel disease (10).
The NLRP3 inflammasome
NLRP3 is part of one of the best-studied inflammasome
complexes. It consists of the NLRP3 scaffold, the ASC
adaptor, and procaspase-1 (3). Two steps are required to
activate the NLRP3 inflammasome (25). The first step is
initiated by microbial ligands or endogenous cytokines
and is needed to induce upregulation of NLRP3 protein
expression (2, 40). NF-rB activation and reactive oxygen
species (ROS) are required for this step. The second step
is activation of NLRP3 by microbial stimuli or endo-
genous molecules (25). NLRP3 is activated by several
microbial-derived ligands, including toxins (20, 37). The
endogenous signals triggering NLRP3 activation include
the danger signal ATP, fatty acids, particulate matter,
necrosis, and necroptosis (reviewed by Hao et al. (25)).
Also K
efflux, lysosome function, endoplasmic reticu-
lum (ER) stress, intracellular calcium, ubiquitination,
microRNAs, and particularly ROS have been proposed
(reviewed by Abais et al. (19)) (Fig. 2). ROS may serve
a ‘kindling’ or triggering factor for activation of the
NLRP3 inflammasome as well as ‘bonfire’ or ‘effector’
molecules leading to pathological processes (19).
In monocytes and dendritic cells, TLR stimulation is
adequate to induce caspase-1 activation and IL-1bproduc-
tion but not in macrophages (reviewed by Hao et al. (25)).
Fig. 1. Major inflammasomes with known stimulators. In NLPR1 muramyl dipeptide and Bacillus anthracis lethal toxin can
directly cause caspase-5 processing. NLRC4 activation is mostly related to components of Gram-negative bacteria. In AIM2,
double-stranded DNA (dsDNA) binds to the HIN200 domain and requires ASC for processing of caspase-1. Also, NLPR3
requires ASC and caspase-1. It is activated in response to both exogenous and endogenous danger signals. (From ref. 19 with
permission.)
Modulation of inflammasome activity
Citation: Journal of Oral Microbiology 2016, 8: 30385 - http://dx.doi.org/10.3402/jom.v8.30385 3
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In human monocytes, TLR stimulation promotes extra-
cellular release of ATP which in turn stimulates the
purinergic receptor P2X
7
needed for activation of the
NLRP3 inflammasome. In dendritic cells, however, NLRP3
activation is independent of P2X
7
(41). This implies that
in some cell types TLR can activate the NLRP3 in-
flammasome independent of extracellular mediators such
as ATP. All NLRP3s have a unique sensor protein of the
NLR or the PYHIN superfamily. These proteins seem to
possess numerous mechanisms for sensing bacteria and
initiating immune mechanisms (10).
The AIM2 inflammasome
AIM2 is a cytosolic binding receptor for double-stranded
DNA (1). It is known to form an inflammasome and
activate caspase-1 when bacteria and viruses are present
(4244). AIM2 consists of an N-terminal pyrin domain
and a C-terminal DNA-binding HIN200 domain. It is
the only known HIN200 domain-containing protein with
capacity to mature IL-1band IL-18 through interactions
with ASC and caspase-1 (42). The AIM2 inflammasome
is particularly important in the defense against intracel-
lular bacterial and viral pathogens (43, 44).
Fig. 2. Activation of the NLRP3 inflammasome as a two-step mechanism. Primary signals come from activation of toll-like
receptors (TLRs) which are responsible for the upregulation of NLRP3 and pro-interleukin-1b(IL-1b)inanNF-kB-dependent
manner. Secondary signals come from several pathways: K
efflux via P2X
7
receptor activation via ATPe coupling, endoplasmic
reticulum (ER) stress, mitochondrial dysfunction, NADPH oxidase, frustrated phagocytosis, and lysosomal rupture pathways. All
these primary and secondary signals converge in the production of reactive oxygen species (ROS). (From ref. 19 with permission.)
Ingar Olsen and O
¨zlem Yilmaz
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P. gingivalis
Synergy
Several opportunistic pathogens have been shown to
develop different mechanisms to inhibit inflammasome
activation and function. Similarly, P. gingivalis, a proposed
keystone organism in chronic periodontitis (17), has been
found to manipulate innate immunity via a number of
mechanisms (18). This bacterium has been postulated to
suppress inflammasome activation as a mechanism for its
low immunostimulatory activity and pathogenic synergy
with other periodontal bacteria that are shown to be more
immunogenic (45). P. gingivalis can also suppress inflam-
masome activation by Fusobacterium nucleatum and this
may be a contribution from P. gingivalis to the synergy
between the two periodontal bacteria (4649). This
specific inhibition appears to affect IL-1band IL-18
processing and cell death in macrophages of both man
and mouse. While F. nucleatum activated IL-1bproces-
sing through the NLRP3 inflammasome, P. gingivalis
mediated repression was not related to lowered levels of
inflammasome components (45). P. gingivalis infection
also influences endocytosis by preferentially suppressing
endocytic pathways toward inflammasome activation.
This represents a new mechanism of pathogen-mediated
inflammasome inhibition (45). It should also be noted
that although P. gingivalis inhibits an activation path-
way that can kill the microrganism; this may not be the
integral part of a general immune suppression strategy as
P. gingivalis harnesses acute sustained inflammation that
is relatively harmless to the bacterium. Indeed, period-
ontitis-associated bacteria could benefit from a nutrition-
ally favorable inflammatory environment created by P.
gingivalis (50).
Nucleoside-diphosphate kinase
P. gingivalis uses its extracellularly secreted nucleoside-
diphosphate kinase homologue (NDK) (51) to inhibit
innate immune responses due to stimulation by extracel-
lular ATP (ATPe) (52). ATPe acts as a danger signal that
can alert the immune system about a present infection.
ATPe binds to P2X
7
receptors (see below) and activates an
inflammasome and caspase-1. Infection of gingival epithe-
lial cells (GECs) resulted in inhibition of ATP-induced
caspase-1 activation (52). ndk-deficient P. gingivalis was
less effective in limiting ATP-mediated caspase-1 activa-
tion and secretion of IL-1bfrom infected cells. NDK
therefore seems to play an important role in inhibiting
P2X
7
-dependent inflammasome activation. The conse-
quent inhibition of P2X
7
-mediated apoptosis and exten-
sion of the viability of GECs could make P. gingivalis
survive for extended periods of time in the gingival
epithelium and contribute to disease when other host and
bacterial factors participate in tissue destruction. NDK
also reduced ATPe-mediated plasma membrane permea-
bilization of host cells in a dose-dependent manner (53).
In GECs, NDK of P. gingivalis promoted intracellular
persistence by inhibiting ATP-induced ROS via P2X
7
receptor/NADPH oxidase signaling (54). This implied
that GECs produced significant amounts of ROS in
response to ATPe and that this depended on P2X
7
-
signaling coupled with membrane-bound NADP oxidase
and the mitochondrial respiratory chain. This novel sig-
naling cascade may contribute to successful tissue per-
sistence of this major pathogen.
Also, the secreted multi-functional effector molecule,
NDK from P. gingivalis attenuated release of the high-
mobility group protein B1 (HMGB1) (52). HMGB1 is a
pro-inflammatory danger signal associated with chroma-
tin in healthy cells. Lack of NDK reduced significantly
the inhibition of ATP-dependent inflammasome activa-
tion and the release of pro-inflammatory cytokines in
GECs (52). The findings suggested that NDK could play
a significant role in the inhibition of P2X
7
-dependent
inflammasome activation and HMGB1-release from
infected GECs.
The P2X
7
and P2X
4
receptors
The P2X
7
purinergic cell surface receptor, which is
expressed on a variety of immune cells, including macro-
phages, functions as a second signal for assembly of the
NLRP3 inflammasome (55). Purinergic signaling is
essential for the release of IL-1bfrom cells infected
with P. gingivalis (56). In macrophages P2X
7
has a dual
role, as it was critical not only for ATPe-induced IL-1b
secretion in vitro but also for intracellular pro-IL-1b
processing (57). These findings also applied to the in vivo
situation since the P2X
7
receptor expression was upregu-
lated in a P. gingivalis oral infection model. Further, the
P2X
7
receptor and NLRP3 transcription were found to
be modulated in human chronic periodontitis (57), sug-
gesting that the P2X
7
receptor also has a role in period-
ontal immunopathogenesis. The ability of P. gingivalis to
modulate ATP-/P2X
7
-signaling, to secrete NDK during
infection in primary GECs, and its expression of other
virulence factors, for example, gingipains and fimbriae,
and promotion of peripheral artery disease (PAD) may
link this bacterium to periodontitis and other systemic
diseases such as rheumatoid arthritis, diabetes, obesity,
multiple sclerosis, and pancreatic and kidney diseases
(58, 59). Interestingly, gingipains may also affect inflam-
masome activation. Jung et al. (60) recently showed that
the simultaneous protease action of Kgp and Rgps atten-
uates the caspase-1 activating potential of P. gingivalis in
macrophages.
It is well known that infection of GECs with P.
gingivalis requires an exogenous danger signal such as
ATPe for activation of an inflammasome and caspase-1.
This again will induce secretion of IL-1b. Generation
Modulation of inflammasome activity
Citation: Journal of Oral Microbiology 2016, 8: 30385 - http://dx.doi.org/10.3402/jom.v8.30385 5
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of ROS is also stimulated by ATPe. However, the
mechanism of ROS generation and the role of purinergic
receptors in inflammasome activation were not very clear
until Hung et al. (61) demonstrated that the purinergic
receptor P2X
4
is assembled with P2X
7
and its associated
pore, pannexin-1. ROS production was induced by ATPe
through a complex containing P2X
4
,P2X
7
, and pannexin-1.
The P2X
7
-mediated ROS production can activate the
NLPR3 inflammasome and caspase-1. Activation by
P2X
4
alone induced generation of ROS but not inflam-
masome activation. Depletion or inhibition of P2X
4
,
P2X
7
, or the pannexin complex markedly blocked IL-1b
secretion in P. gingivalis-infected GECs after ATPe treat-
ment (61). Accordingly, ROS is produced by stimulation
of the P2X
4
/P2X
7
/pannexin-1 complex. This also means
that P2X
4
acts as a positive regulator of inflammasome
activation during infection with P. gingivalis.
A2a adenosine receptor
Danger signals (DS) are molecules like adenosine that
exert extracellular signaling derived from autocrine and/
or paracrine secretions during inflammation and chronic
diseases. Adenosine, which is a metabolite of ATP, has
so far been little appreciated as a component of the innate
immune system (62). ATPe is produced through a num-
ber of enzymatic reactions in normal, stressed, or infected
tissues (63). It has recently been shown that P. gingivalis
can use A2A adenosine receptorcoupled DS adenosine
signaling as a means to proliferate and survive in primary
GECs, possibly by down-regulating the pro-inflammatory
response (62). P. gingivalis reduces extracellular nucleo-
tide concentrations of ATP and thereby acts as a gen-
erator of adenosine which stimulates the bacterium’s
growth in primary GECs. This may be another anti-
inflammatory immune response exerted by P. gingivalis to
promote its survival in the oral mucosa.
Phosphatidylserine
During Chlamydia infection of human epithelial, en-
dothelial, granulocyte, or monocytic cells, phosphatidyl-
serine (PS) is translocated from the inner to the outer
leaflet and becomes exposed to the external side of the
cell (64). PS exposure is an early marker of apoptosis
associated with pro-inflammatory triggering of comple-
ment activation (65, 66). Yilmaz et al. (67) found that P.
gingivalis after establishing itself in the nutritionally rich
cytosol of the host cell can protect the infected cell from
the host immune defense by reducing the inflammatory
response after inducing transient externalization of PS.
This would allow multiplication of P. gingivalis inside
cells while protecting them from cytotoxic reactions
of the immune system. It was also suggested that the
bacterium blocks mitochondrion-dependent apoptosis to
upkeep its intracellular lifestyle. This may permit success-
ful spreading of P. gingivalis to adjacent and deeper host
tissues.
Inflammasome activity in P. gingivalisinduced
periodontal disease and defense
GECs are important parts of the immune response to
periodontal bacteria. They express a functional NLRP3
inflammasome (68). Much higher levels of inflamma-
some components were found in the gingival tissues from
patients with chronic periodontitis than from healthy
controls (14). It therefore seems reasonable to consider
the inflammasome as an operational part of innate im-
munity against periodontitis.
While supragingival biofilm, causing gingivitis, in-
creased the expression of caspase-1, ASC, AIM2, IL-
1b, and IL-18 in gingival fibroblasts, subgingival biofilm,
promoting periodontitis, enhanced caspase-1, ASC,
AIM2, IL-1b, and IL-18 gene expression at lower
concentrations, followed by their downregulation at
higher concentrations (69). The authors proposed that
high concentrations of bacterial virulence factors in sites
with affluent immune mechanisms such as the biofilm-
tissue interface can down-regulate host defense barriers,
while the lower bacterial concentrations deeper in period-
ontal tissues can have a stimulatory effect on inflamma-
tory responses.
When using a 10-species subgingival biofilm with P.
gingivalis present, Belibasakis et al. (70) found a reduc-
tion in NLRP3 and IL-1bexpression in human gingival
fibroblasts after challenge for 6 h. The AIM2 expression
was not affected. After exclusion of P. gingivalis from the
biofilm, a partial rescue of NLRP3 and IL-1b-expression
occurred. It was suggested that subgingival biofilms
down-regulate NLRP3 and IL-1bexpression partly due
to P. gingivalis and that this dampening of the host innate
immune responses may favor persistence and survival of
biofilm species in periodontal tissues.
It has been recently shown that fimbriae of P. gingivalis
inhibit ATPe-induced IL-1bsecretion through the
P2X
7
receptor in macrophages (56). Ramos-Junior
et al. (57), however, found that NLRP3 is necessary for
ATPe-induced IL-1bsecretion as well as for caspase-1
activation irrespective of P. gingivalis fimbriae. Although
IL-1bsecretion from P. gingivalisinfected macrophages
depended on NLRP3, its adaptor protein ASC, or
caspase-1, the cleavage of intracellular IL-1bto the
mature form occurred independently of NLRP3, its
adaptor protein ASC, or caspase-1.
P. gingivalis dampened ATPe-induced IL-1bsecretion
in macrophages by means of its fimbriae in a purinergic
P2X
7
receptor-dependent manner (56). In this study,
the immune subversion of P. gingivalis was connected
with the ability of fimbriae to reduce ATPe-induced
macrophage secretion of IL-1bvia P2X
7
activation. The
authors held that this could be another molecular action
of subversion of the immune system by P. gingivalis.
In THP-1 (leukemic monocytic) cells, P. gingivalis
induced IL-1bsecretion and inflammatory cell death
Ingar Olsen and O
¨zlem Yilmaz
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through activation of caspase-1 (14). IL-1bsecretion and
pyroptic cell death required both NLRP3 and AIM2
inflammasome activation via TLR2 and TLR4 signaling.
The activation of the former was mediated by ATP
release, the P2X
7
receptor, and lysosomal damage (14).
These authors also suggested that P. gingivalisinduced
NLRP3 activation depends on ATP release, K
efflux,
and cathepsin B.
Underacylated lipopolysaccharide
P. gingivalis can use lipid A phosphatase to alter the lipid
A composition of its lipopolysaccharide (LPS). This
organism may therefore modulate the immune response
by expressing an underacylated LPS (7173). This LPS
could bind but not activate caspase-11 (human caspase-
4,5) which results in host cell lysis and reduces sur-
vival of bacteria (72, 74). Caspase-4 can be of specific
importance to mucosal immunity since caspase-4 expres-
sion level and ability to become activated as a response
to infection differ markedly from that of caspase-5.
Caspase-4 has been suggested to provide key non-
redundant discernment into rapid sensing and clearance
of bacteria at mucosal tissues (21).
Atherosclerosis
There is mounting evidence that P. gingivalis can invade
cardiovascular cells and tissues causing inflammation
(75). The NLRP3 inflammasome has been suggested to
have an important role in developing vascular inflam-
mation and atherosclerosis (76, 77). In hyperlipidemic
animals, P. gingivalis accelerated atherosclerosis (78). Wild-
type challenge of apolipoprotein Edeficient, sponta-
neously hyperlipidemic (Apoe
shl
) mice with P. gingivalis
increased IL-1b, IL-18, and TNF-aproduction in peri-
toneal macrophages and gingival or aortic gene expres-
sion of the NOD-like receptor family, NLRP3, IL-1b,
pro-IL-18 and pro-caspase-1 (78). Fimbriae were found to
bring increased tissue invasiveness and pro-inflammatory
ability to P. gingivalis. It was also demonstrated that
P. gingivalis activates innate immune cells through the
NLRP3 inflammasome compared with a KDP136 (gingipain-
null) or a KDP150 (FimA-deficient) mutant.
Inflammation-related induction of AIM2 in vascular
cells and atherosclerotic lesions has suggested a role for
AIM2 in vascular pathogenesis where increased AIM2
expression was seen around the necrotic core of athero-
sclerotic carotid lesions and in the vasa vasorum of
neovasculature of aortic aneurysms (79). The NLRP3
inflammasome and AIM2 may thus have important roles
in both P. gingivalisinduced periodontal disease and
atherosclerosis through sustained inflammation.
Recently, the CD36/scavenger receptor (SR)-B2 was sug-
gested to play a role at multiple points in P. gingivalis
mediated enhanced atherosclerosis in a mouse model
(80). The study suggested that activation of the inflam-
masome by P. gingivalis is mediated by CD36/SR-B2 and
TLR2 which cause systemic release of pro-atherosclerotic
IL-1band macrophage pyroptosis. Systemic IL-1bacti-
vates vascular macrophages naı
¨ve to P. gingivalis to
secrete IL-1band promotes CD36-mediated uptake of
oxLDL and increased formation of foam cells. The
presence of oxLDL could inhibit P. gingivalis/P. gingivalis
LPS-inflammasome activation and pyroptosis, which
would allow greater atherosclerotic plaque to develop.
TLR-CD36-/SR-B2-mediated IL-1bgeneration may thus
be important to increase atherosclerotic lesions. Of note
is also that cytoplasmic LPS sensing in human cells
activates the non-canonical caspase-4-dependent inflam-
masome. This is a new mechanism of inflammasome
activation where direct LPS-binding results in caspase
oligomerization and activation leading to induction of
IL-1bsecretion and pyroptosis (81).
Alzheimer’s disease
P. gingivalis may be an important pathogen in Alzhei-
mer’s disease (AD) contributing to brain inflammation
(82). NLRP3 has been reported in microglial cells that
responded to infection and initiation of neuro-degeneration
in an Alzheimer’s disease model (83). Furthermore,
TLR2 and NLRP3 were recently found to co-operate to
recognize a functional bacterial amyloid, curli fibers, in
the plaques of brains in AD patients (84). Heneka et al.
(85) found a strongly activated caspase-1 expression in
human mild cognitive impairment and AD brains which
suggested a role for the inflammasome in brain degen-
erative disease. AD brain deposits activated the NLRP3
inflammasome in microglial cells in vitro and in vivo
which could lead to progression of AD (8588). This
suggested a role for the inflammasome in this neurode-
generative disease.
Non-alcoholic steatohepatitis
P. gingivalis seems to be a critical risk factor for pro-
gression of non-alcoholic steatohepatitis (NASH) through
upregulation of the P. gingivalis-LPS-TLR2-pathway and
by stimulating inflammasomes (89). These authors found
that P. gingivalis exacerbated steatohepatitis induced by
diet via induction of inflammasomes and inflammatory
cytokines in the liver of mice. P. gingivalis was also
demonstrated for the first time in the liver of NASH
patients. It was suggested that dental infection with P.
gingivalis promotes progression of NASH.
Squamous cell carcinoma
A close association has been found between P. gingivalis
and squamous cell carcinoma (90). The fact that P.
gingivalis modulates ATP-/P2X
7
-signaling; secretes the
anti-apoptotic enzyme, NDK, during infection of primary
OECs; and expresses other virulence factors, such as
fimbriae, gingipains, and PAD, that may be potential
etiologic links to orodigestive cancers and other chronic
diseases (58, 59). Of note is also that IL-1bpromotes
Modulation of inflammasome activity
Citation: Journal of Oral Microbiology 2016, 8: 30385 - http://dx.doi.org/10.3402/jom.v8.30385 7
(page number not for citation purpose)
malignant transformation of tumor aggressiveness in oral
cancer (91).
Rheumatoid arthritis
Periodontitis is more prevalent in patients with rheuma-
toid arthritis (RA) than in those without (92). RA is also
prevalent in patients with periodontitis (93). Besides,
there is improvement in RA after periodontal treatment
and in periodontitis after treatment of RA (94, 95).
Furthermore, DNA from a variety of oral bacteria,
including P. gingivalis, has been detected in synovial fluid
of active RA (96). Bostanci et al. (97) found a positive
correlation between NLRP3 and expression of IL-1band
IL-18 in periodontitis, and upregulated levels of NLRP3,
AIM2, and caspase-1 have been detected in gingival
tissues of patients with periodontitis (14). Probably, P.
gingivalis manipulates the host inflammatory responses
to be able to survive and prevail within infected period-
ontal tissues (97), which it may achieve by limiting or
controlling the activation of the NLRP3 inflammasome.
It seems reasonable to expect similar effects in distant
sites, for example, in the joints of rheumatic patients
where P. gingivalis can be present.
Concluding remarks
Inflammasomes represent a relatively new concept in
innate immunity. There is great variation in inflamma-
somes and the mechanisms by which they detect and resist
pathogens. Many interactions between inflammasomes
and the innate immune system are still unknown. It is
becoming clear that the inflammasome and its constitu-
ents are likely crucial in the initiation of periodontal
disease and several chronic systemic diseases associated
with periodontitis. Nevertheless, it may be difficult to
intervene in inflammasome actions for the purpose of
treating disease since interfering with key parts in the
complex may have serious local and systemic effects. The
ubiquitous distribution and importance of inflammasome
activation in many peripheral processes adds to this
limitation. There are still not fully understood roles of all
players in the inflammasome complex where anti-inflam-
matory therapies might not be sufficient to treat the roots
of the disease. Knowledge about inflammasomes has
mainly been retrieved from murine systems. The applic-
ability of some of these results to human cells is unclear
because gene products differ between species, and the
specificity of ligands is not always conserved. P. gingivalis
has a number of ways for suppressing innate immunity
and inflammasome activity. Although this subversion
probably is important for periodontitis and some related
systemic diseases, it remains to see if other parts of the oral
microbiota can behave in a similar way and if this sub-
version can affect players other than F. nucleatum in the
dental biofilm. Efforts should also be made to see how
inflammasomes can affect the ecology of the dental plaque
microbiota.
Conflict of interest and funding
There is no conflict of interest in the present study for any
of the authors. IO acknowledges funding through a grant
from the European Commission (FP7-HEALTH-306029
‘TRIGGER’) and O
¨Y acknowledges funding through a
NIDCR grant R01DE016593.
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Modulation of inflammasome activity
Citation: Journal of Oral Microbiology 2016, 8: 30385 - http://dx.doi.org/10.3402/jom.v8.30385 11
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... IL-1 β incites the expression of genes that regulate fever, pain threshold, vasodilation, and hypotension, and its reception results in an endothelial cell response that facilitates immune cell infiltration into infected or damaged tissues (37). Inflammasome formations is a response to cellular infections, cellular stress or damage of the cells (38). Particularly, IL-1 β and IL-18 activities is central to the immune response of the host in periodontitis (39). ...
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Periodontitis is an inflammatory condition associated with bacterial overgrowth, and Gram-negative bacteria within the periodontal pockets have been shown to raise inflammatory markers. The NLRP3inflammasome is a vital elements of the innate immune system that activates and secretes pro-inflammatory cytokines in response to microbial (viral, bacterial. etc.) infections. Chronic inflammation brought on by host immunological responses is a common feature of periodontitis. The purpose of this review is to outline the function of the NLRP3 inflammasome in periodontal disease.
... To date, several inflammasomes have been described. NLRP3, as the most studied inflammasome, is activated by the infected pathogens and releasing of endogenous danger signals and then drives pathological inflammation in periodontitis [38][39][40]. In this review, we describe the recent progress and our current understanding of NLRP3 inflammasome pathogenesis in periodontitis. ...
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Periodontitis is a chronic inflammatory disease that affects tooth-supporting tissues and even leads to tooth loss. NLRP3 inflammasomes play a critical role in periodontitis pathogenesis. Aberrant activation or overexpression of NLRP3 inflammasomes in cellular players, including osteoclasts, osteoblasts, periodontal ligament fibroblasts, and leukocytes often contributes to cellular dysfunction and environment abnormality, thus resulting in the disorganization of ligament and alveolar bone. In this review, we mainly focus on the negative regulation of NLRP3 inflammasome in periodontitis and highlight the importance of NLRP3 inflammasome as a candidate therapeutic target in periodontitis treatment. Then we elucidate the development status of NLRP3 inflammasome inhibitors and show their application potential for treating periodontitis. In summary, this review reveals the recent progress and perspectives of NLRP3 inflammasome and the therapeutic potential of NLRP3 inflammasome inhibitors in periodontitis.
... Typical inflammasomes are constructed of pro-caspase-1, nucleotide-binding domain (NBD), and leucine-rich repeats (LRRs), called the NBD-LRR (NLR) superfamily that is responsible for the recognition of pathogen-associated molecular patterns (PAMPs) or other signals (65) and adapter molecule apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) (Figure 2). Among these, caspase-1 cleaves pro-IL-1b and pro-IL-18, it also mediates their maturation and excretion (66). Inflammasome activation consists of two steps, an initial "cell priming" and a second "triggering" event, resulting in the proteolytic maturation and secretion of IL-1b (2). ...
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Porphyromonas gingivalis (P. gingivalis) is a Gram-negative anaerobic pathogen that is involved in the pathogenesis of periodontitis and systemic diseases. P. gingivalis has recently been detected in rheumatoid arthritis (RA), cardiovascular disease, and tumors, as well as Alzheimer’s disease (AD), and the presence of P. gingivalis in these diseases are correlated with poor prognosis. Macrophages are major innate immune cells which modulate immune responses against pathogens, however, multiple bacteria have evolved abilities to evade or even subvert the macrophages’ immune response, in which subsequently promote the diseases’ initiation and progression. P. gingivalis as a keystone pathogen of periodontitis has received increasing attention for the onset and development of systemic diseases. P. gingivalis induces macrophage polarization and inflammasome activation. It also causes immune response evasion which plays important roles in promoting inflammatory diseases, autoimmune diseases, and tumor development. In this review, we summarize recent discoveries on the interaction of P. gingivalis and macrophages in relevant disease development and progression, such as periodontitis, atherosclerosis, RA, AD, and cancers, aiming to provide an in-depth mechanistic understanding of this interaction and potential therapeutic strategies.
... In the context of periodontitis, a number of recent in vitro and animal experimental studies have also documented the activation of GSDMD and NLRP3 inflammasome pathways, including that by P. gingivalis lipopolysaccharide (LPS) stimulation (18)(19)(20)(21)(22)(23). P. gingivalis induced the activation of pyroptosis-related NLRP3 inflammasome, which is also implicated in atherosclerosis associated with periodontitis (24,25). The inhibition of NLRP3 inflammasome in periodontitis is also documented as a mechanism of immune evasion (26,27). ...
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Aim: To identify pyroptosis-related genes (PRGs), their functional immune characteristics, and distinct pyroptosis-related clusters in periodontitis. Methods: Differentially expressed (DE)-PRGs were determined by merging the expression profiles of GSE10334, GSE16134, and PRGs obtained from previous literatures and Molecular Signatures Database (MSigDB). Least Absolute Shrinkage and Selection Operator (LASSO) regression was applied to screen the prognostic PRGs and develop a prognostic model. Consensus clustering was applied to determine the pyroptosis-related clusters. Functional analysis and single-sample gene set enrichment analysis (ssGSEA) were performed to explore the biological characteristics and immune activities of the clusters. The hub pyroptosis-related modules were defined using weighted correlation network analysis (WGCNA). Results: Of the 26 periodontitis related DE-PRGs, the highest positive relevance was for High-Mobility Group Box 1 (HMGB1) and SR-Related CTD Associated Factor 11 (SCAF11). A 14 PRGs-based signature was developed through LASSO model, In addition, three pyroptosis-related clusters were obtained based on the 14 prognostic PRGs. Caspase 3 (CASP3), Granzyme B (GZMB), Interleukin 1 Alpha (IL1A), IL1Beta (B), IL6, Phospholipase C Gamma 1 (PLCG1) and PYD And CARD Domain Containing (PYCARD) were dysregulated in the three clusters. Distinct biological functions and immune activities including Human Leukocyte Antigen (HLA) gene expression, immune cell infiltration, and immune pathway activities were identified in the three pyroptosis-related clusters of periodontitis. Furthermore, the pink module associated with endoplasmic stress related functions, was found to be correlated with cluster 2, and was suggested as the hub pyroptosis-related module. Conclusion: The study identified 14 key pyroptosis-related genes, three distinct pyroptosis-related clusters, and one pyroptosis related gene module, describing several molecular aspects of pyroptosis in the pathogenesis and immune micro-environment regulation of periodontitis and also highlighted functional heterogeneity in pyroptosis-related mechanisms.
... Our previous studies have demonstrated that SGK1 promotes macrophage polarization to M2 and restrains secretion of TLR-mediated inflammatory cytokines (22,27,29). P. gingivalis is a key pathogenic bacterium that triggers periodontitis and was associated with multiple inflammatory diseases, such as arthritis, atherosclerosis, and Alzheimer's disease (41)(42)(43). To examine if SGK1 also functions in P. gingivalis-induced inflammation and elucidate the molecular mechanisms involved, we first examined activation of SGK1 in response to the challenge of P. gingivalis in different innate cells. ...
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SGK1 is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanism involved remains elusive, especially in the oral bacteria-induced inflammatory milieu. In this study, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of TNF receptor-associated factor (TRAF) 3, and thereby suppressing NF-κB signaling. We show pharmacological inhibition of SGK1 significantly enhances the production of pro-inflammatory cytokines including TNFα, IL-6, IL-1β, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 knockout mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression, but failed to alter activation of MAPK signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates phosphorylation of TRAF2, which leads to TRAF3 degradation in a proteasome-dependent manner. Importantly, we show siRNA-mediated traf3 silencing or overexpression of c-Jun mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. Additionally, using a P. gingivalis infection-induced periodontal bone loss model, we found inhibition of SGK1 modulates expression of TRAF3 and c-Jun, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun/NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets relevant to inflammatory diseases that could extend beyond the oral cavity.
... While they have largely been ignored as potential primary influencers of liver abscess syndrome in cattle, species of Bacteroides and Porphyromonas are commonly found in anaerobic infections and have been extensively implicated in non-liver abscess formation in humans (Gibson et al., 1998;Murakami et al., 2001;Van der Cruyssen et al., 2017). They also contain potent virulence factors and are capable of activating the inflammosome (Olsen and Yilmaz, 2016), making it feasible that they could significantly contribute to LA formation. ...
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Liver abscesses (LAs) are extremely prevalent in cattle and result in significant economic losses due to liver condemnation, decreased growth and production, and lower carcass quality. LAs are commonly attributed to the transition to diets high in rapidly fermentable starch which results in rumen epithelial inflammation that allows pathogenic bacteria to gain entry to liver through transport via the hepatic portal vein. The most common intervention for LAs is the inclusion of antibiotics in feedlot diets, under the supervision of a veterinarian; this treatment is associated with reduced occurrence of LAs in this and other studies. Here, through the largest LA 16S rRNA gene sequencing study to date, we demonstrate that the inclusion of tylosin and antibiotic alternatives (the essential oil limonene and Saccharomyces cerevisiae fermentation product) had little impact on LA microbial community composition. Importantly, members of Bacteroidetes (Bacteroides spp. and Porphyromonas spp.) were identified as the dominant taxa in conjunction with low proportions of Fusobacteria in nearly a quarter (61/259) of all LA communities analyzed in this study. The relative abundances of the phyla Fusobacteria and Bacteroidetes had a strongly negative correlation, and LA microbial communities rarely contained high abundances of both of these dominant phyla. Further, based on the presence of taxa discriminant of Bacteroidetes-dominated LAs within over 400 bovine gut communities, we provide evidence suggestive of Bacteroidetes-dominated abscess communities originating in more distal portions of the bovine gut. Together, these findings suggest that some LA microbial communities may originate from portions of the gut other than the rumen.
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Mycoplasma gallisepticum (MG) is a pathogenic microorganism that causes chronic respiratory disease (CRD). MG infection has a serious negative impact on the poultry industry. Andrographolide (AG) is known to regulate immune responses, antimicrobial infections, and anti-inflammatory responses. However, the underlying molecular mechanisms of AG action in MG-infected chickens remain unclear. Hence, we constructed models of MG infection by using chickens and chicken macrophage-like (HD11) cells in vivo and in vitro, respectively. The results showed that AG significantly inhibited the mRNA and protein expression of the toxic adhesion protein pMGA1.2 in vivo and in vitro. Meanwhile, AG treatment significantly decreased the mRNA expression of pro-inflammatory such as interleukin-6 (IL-6) and interleukin- 1β (IL-1β), and increased the mRNA expression of an anti-inflammatory such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) in vivo and in vitro. Furthermore, AG treatment down-regulated inflammasome NLRP3 and apoptosis genes caspase3 and caspase9, and up-regulated autophagy protein light chain 3 (LC3) by regulating the PI3K/Akt signaling pathway in vitro. Our results suggest that AG can reduce the expression of NLRP3 and alleviate the inflammatory response from MG infection by inducing autophagy, probably by modulating PI3K/Akt signaling pathway. This study demonstrates that AG can be used as a specific target to prevent and treat MG infection effectively.
Chapter
The gut microbiome is critical for overall human health. Many factors can disturb the gut microbiome and create dysbiosis. Both the mucous layer and the gut epithelial lining, which collectively serve as the gastrointestinal mucosal border, can be structurally degraded by gut dysbiosis. Increased intestinal permeability and compromised host resistance can ensue. Since all mucosal tissues in the body participate in cross-talk, other organ systems can be affected according to genetic predispositions and an increase in systemic chronic inflammation. The manifestation of named chronic diseases is the ultimate outcome. In this case, symptoms of various chronic diseases must be addressed. Specifically, periodontal disease, which is a chronic disease, must be understood and treated appropriately because it can become a second nidus of infection along with the primary nidus emanating from gut dysbiosis. However, the mystery of treatment does not only require medical intervention, although medical treatment for acute disease is critical. Essential treatment for this scenario usually includes a dietary and lifestyle solution to restore healthy gastrointestinal tract function. Unraveling the mystery of treatment involves introducing viable probiotics and prebiotics; removing elements of an inflammatory diet; providing necessary nutrients and nourishment for cellular function, eliminating toxic elements and chemicals; and creating a healthy lifestyle of stress reduction, restorative sleep, and efficient exercise.
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Subject. Parallels in the pathogenesis of two severe diseases of the modern era, osteoporosis and periodontitis. Objectives. To review domestic and international research on pathogenetic relationship between osteoporosis and periodontal pathology. Methodology. In topic generalization, the review of publications (since 2016) available on PubMed, eLIBRARY, Web of Science, Scopus by keywords is made. Conclusion. Research studies have shown that bone tissue in the orofacial region, though lesser than bones of axial skeleton, tends to develop osteoporosis. That is why patients with osteoporosis are recommended regular periodontal maintenance visits, especially when periodontal disease is diagnosed at the appointment or during the complex rehabilitation program for patients in osteoplastic and maxillofacial reconstructive surgery. Future controlled longitudinal studies may be useful in research of this relationship based on the features of osteogenesis in the both diseases. This literature review helps to develop modern views of pathogenetic relationship between osteoporosis and periodontitis, evaluate general risk factors, promote understanding of tools in the diagnostic process and interpretation of results. Moreover, with the help of this paper by knowing the cell and molecular structure of bone tissue and mechanisms of bone remodeling a dental practitioner can arrange a personalized follow-up strategy for patients in the risk group for the above two diseases timely engaging general practitioners in interdisciplinary and complex therapy of patients with periodontal pathology.
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Recently, the focus of murine caspase-11 and human orthologs caspase-4, -5 research has been on their novel function to induce noncanonical inflammasome activation in direct response to Gram-negative bacterial infection. On the other hand, a new role in anti-bacterial autophagy has been attributed to caspase-11, -4 and -5, which currently stands largely unexplored. In this review, we connect lately emerged evidence that suggests these caspases have a key role in anti-bacterial autophagy and discuss the growing implications of a danger molecule-extracellular ATP-and NADPH oxidase-mediated ROS generation as novel inducers of human caspase-4, -5 signaling during infection. We also highlight the adeptness of persistent pathogens like Porphyromonas gingivalis, a Gram-negative anaerobe and successful colonizer of oral mucosa, to potentially interfere with the activated caspase-4 pathway and autophagy. While, the ability of caspase-4, -5 to promote autophagolysosomal fusion is not well understood, the abundance of caspase-4 in skin and other mucosal epithelial cells implies an important role for caspase-4 in mucosal defense, supporting the view that caspase-4, -5 may play a non-redundant part in innate immunity. Thus, this review will join the currently disconnected cutting-edge research thereby proposing a working model for regulation of caspase-4, -5 in pathogen elimination via cellular-trafficking.
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The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease.
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Alzheimer's disease (AD) is a scourge of longevity that will drain enormous resources from public health budgets in the future. Currently, there is no diagnostic biomarker and/or treatment for this most common form of dementia in humans. AD can be of early familial-onset or sporadic with a late-onset. Apart from the two main hallmarks, amyloid-beta and neurofibrillary tangles, inflammation is a characteristic feature of AD neuropathology. Inflammation may be caused by a local central nervous system insult and/or by peripheral infections. Numerous microorganisms are suspected in AD brains ranging from bacteria (mainly oral and non-oral Treponema species), viruses (herpes simplex type I), and yeasts (Candida species). A causal relationship between periodontal pathogens and non-oral Treponema species of bacteria has been proposed via the amyloid-beta and inflammatory links. Periodontitis constitutes a peripheral oral infection that can provide the brain with intact bacteria and virulence factors and inflammatory mediators due to daily, transient bacteremias. If and when genetic risk factors meet environmental risk factors in the brain, disease is expressed, in which neurocognition may be impacted, leading to the development of dementia. To achieve the goal of finding a diagnostic biomarker and possible prophylactic treatment for AD, there is an initial need to solve the etiological puzzle contributing to its pathogenesis. This review therefore addresses oral infection as the plausible etiology of late-onset AD (LOAD).
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Porphyromonas gingivalis is considered a major pathogen in adult periodontitis and is also associated with multiple systemic diseases, for example, cardiovascular diseases. One of its most important virulence factors is invasion of host cells. The invasion process includes attachment, entry/internalization, trafficking, persistence, and exit. The present review discusses these processes related to P. gingivalis in cardiovascular cells and tissue. Although most P. gingivalis strains invade, the invasion capacity of strains and the mechanisms of invasion including intracellular trafficking among them differ. This is consistent with the fact that there are significant differences in the pathogenicity of P. gingivalis strains. P. gingivalis invasion mechanisms are also dependent on types of host cells. Although much is known about the invasion process of P. gingivalis, we still have little knowledge of its exit mechanisms. Nevertheless, it is intriguing that P. gingivalis can remain viable in human cardiovascular cells and atherosclerotic plaque and later exit and re-enter previously uninfected host cells.
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Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1β and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1β secretion by means of its fimbriae in a purinergic P2X7 receptor-dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1β processing and secretion by P. gingivalis-infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1β secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1β from P. gingivalis-infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1β to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1β secretion but also for intracellular pro-IL-1β processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-γ and IL-17 were detected in draining lymph node cells from P2rx7(-/-) mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis. © International & American Associations for Dental Research 2015.
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There is strong epidemiological association between periodontal disease and cardiovascular disease but underlying mechanisms remain ill-defined. Because the human periodontal disease pathogen, Porphyromonas gingivalis (Pg), interacts with innate immune receptors Toll-like Receptor (TLR) 2 and CD36/scavenger receptor-B2 (SR-B2), we studied how CD36/SR-B2 and TLR pathways promote Pg-mediated atherosclerosis. Western diet fed low density lipoprotein receptor knockout (Ldlr° ) mice infected orally with Pg had a significant increase in lesion burden compared with uninfected controls. This increase was entirely CD36/SR-B2-dependent, as there was no significant change in lesion burden between infected and uninfected Ldlr° mice. Western diet feeding promoted enhanced CD36/SR-B2- dependent IL1β generation and foam cell formation as a result of Pg lipopolysaccharide (PgLPS) exposure. CD36/SR-B2 and TLR2 were necessary for inflammasome activation and optimal IL1β generation, but also resulted in LPS induced lethality (pyroptosis). Modified forms of LDL inhibited Pg-mediated IL1β generation in a CD36/SR-B2-dependent manner and prevented pyroptosis, but promoted foam cell formation. Our data show that Pg infection in the oral cavity can lead to significant TLR2-CD36/SR-B2 dependent IL1β release. In the vessel wall, macrophages encountering systemic release of IL1β, PgLPS and modified LDL have increased lipid uptake, foam cell formation, and release of IL1β, but because pyroptosis is inhibited, this enables macrophage survival and promotes increased plaque development. These studies may explain increased lesion burden as a result of periodontal disease, and suggest strategies for development of therapeutics.
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Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the pro-inflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. Copyright © 2015. Published by Elsevier Masson SAS.
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Associations between oral and systemic health are ancient. Oral opportunistic bacteria, particularly, Porphyromonas gingivalis and Fusobacterium nucleatum, have recently been deviated from their traditional roles as periodontal pathogens and arguably ascended to central players based on their participations in complex co-dependent mechanisms of diverse systemic chronic diseases risk and pathogenesis, including cancers, rheumatoid-arthritis, and diabetes. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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There is extensive evidence that accumulation of mononuclear phagocytes including microglial cells, monocytes, and macrophages at sites of β-amyloid (Aβ) deposition in the brain is an important pathological feature of Alzheimer's disease (AD) and related animal models, and the concentration of these cells clustered around Aβ deposits is several folds higher than in neighboring areas of the brain [1-5]. Microglial cells phagocytose and clear debris, pathogens, and toxins, but they can also be activated to produce inflammatory cytokines, chemokines, and neurotoxins [6]. Over the past decade, the roles of microglial cells in AD have begun to be clarified, and we proposed that these cells play a dichotomous role in the pathogenesis of AD [4, 6-11]. Microglial cells are able to clear soluble and fibrillar Aβ, but continued interactions of these cells with Aβ can lead to an inflammatory response resulting in neurotoxicity. Inflammasomes are inducible high molecular weight protein complexes that are involved in many inflammatory pathological processes. Recently, Aβ was found to activate the NLRP3 inflammasome in microglial cells in vitro and in vivo thereby defining a novel pathway that could lead to progression of AD [12-14]. In this manuscript, we review possible steps leading to Aβ-induced inflammasome activation and discuss how this could contribute to the pathogenesis of AD.
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Inflammasomes are an oligomeric assembly of multiprotein complexes that activate the caspase-1-dependent maturation and the subsequent secretion of inflammatory interleukin-1beta and interleukin-18 cytokines in response to a 'danger signal' in vertebrates. The assessment of their significance continues to grow rapidly as the complex biology of various chronic inflammatory conditions is better dissected. Increasing evidence strongly links inflammasomes and host-derived small 'danger molecule ATP' signaling with the modulation of the host immune response by microbial colonizers as well as with potential altering of the microbiome structure and intermicrobial interactions in the host. All of these factors eventually lead to the destructive chronic inflammatory disease state. In the oral cavity, a highly dynamic and multifaceted interplay takes place between the signaling of endogenous danger molecules and colonizing microbes on the mucosal surfaces. This interaction may redirect the local microenvironment to favor the conversion of the resident microbiome toward pathogenicity. This review outlines the major components of the known inflammasome complexes/mechanisms and highlights their regulation, in particular, by oral microorganisms, in relation to periodontal disease pathology. Better characterization of the cellular and molecular biology of the inflammasome will probably identify important potential therapeutic targets for the treatment and prevention of periodontal disease, as well as for other debilitating chronic diseases. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.