Role of Lipopolysaccharide, Derived from Various Bacterial Species, in Pulpitis—A Systematic Review

Lipopolysaccharide (LPS) is widely used for induction of inflammation in various human tissues, including dental pulp. The purpose of this study was to summarize current medical literature focusing on (1) cell types used by researchers to simulate dental pulp inflammation, (2) LPS variants utilized in experimental settings and how these choices affect the findings. Our study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). We searched for studies reporting outcomes of lipopolysaccharide application on dental pulp cells in vitro using electronic databases: MEDLINE, Web of Science and Scopus. Having gathered data from 115 papers, we aimed to present all known effects LPS has on different cell types present in dental pulp. We focused on specific receptors and particles that are involved in molecular pathways. Our review provides an essential foundation for further research using in vitro models of pulpitis.


Introduction
Pulpitis is an inflammation of the dental pulp-a painful condition mostly caused by Gram-negative bacteria. Lipopolysaccharides, which are components of the Gram-negative bacterial outer membrane, take part in inducing inflammation in the dental pulp. Understanding mechanisms underlying this pathology may help find novel specific treatments.

Cells in Dental Pulp
Dental pulp is a loose connective tissue of ectomesenchymal origin, enclosed by three mineralized materials: dentin, enamel, and cementum [1]. It consists various cells: fibroblasts, odontoblasts, macrophages, mast cells and plasma cells [2]. The odontoblast layer lying on the periphery, at the interface of dentin-pulp complex, is responsible mainly for dentin development [2]. Channels created by odontoblasts extend through dentin and provide nutrient to avascular dentin [2]. Beneath the odontoblast layer lies the cell-poor zone (zone of Weil) [2] and underneath-a cell-rich zone, filled with fibroblasts and undifferentiated mesenchymal cells [2]. At the core, the central pulp contains numerous fibroblasts, vessels and autonomic and sensory nerves, which extend to peripheral layers [2].

Pulpitis
Pulp inflammation is a dynamic and complex process involving neural, vascular and immunological reactions. Microorganisms, dentinal tubules of the teeth, chemical or mechanical irritation, as well as trauma may induce this state [1].
Bacterial infection is the most frequent cause of pulp diseases. Pulp inflammation is primarily the result of cooperation between anaerobic bacteria (such as Porphyromonas
Some studies found alternations in transcriptional factors upon LPS stimulation. One of upregulated proteins is ten-eleven translocation 2 (TET2) DNA methylcytosine dioxygenase [7], responsible for hydroxymethylation of e.g., Myeloid differentiation primary response 88 (MyD88) gene and promoting its expression. LPS stimulation also upregulates pulpitis treatment) [27], showed better viability. The group with MTA and NAC treatment also promoted mineralization [27].
Nel-like molecule type 1 (Nell-1) is a compound used as an osteoinductive factor. Human recombinant Nell-1 attenuates p38 and ERK MAPK pathways and decreases expression of IL-6 and IL-8 in LPS-stimulated cells [31].
Acemannan, a polysaccharide from Aloe vera induces proliferation, differentiation, growth factor and ECM synthesis and formation of mineralized bridge [38]. Huang et al. showed that hDPCs treated with LPS and co-stimulated with odontogenic induction medium had increase ALP activity, DSPP DMP-1 via NF-κB and no change in c-Jun expression [39].
LPS stimulates production of IL-1β, IL-6. After 14 days, cultured cells presented markers of dentinogenesis. Wnt5a, Runx2 and ALP proteins were upregulated, and ALP activity was increased on the 21st day. Wnt5 activation was confirmed by using inhibitor-Box5, which attenuated Runx2 and ALP levels [40].

Fibroblasts
E. coli LPS was used in 7 out of 13 experiments performed on human pulp fibroblasts. Bacteria serotypes were specified in 5 articles-E. coli 055:B5 in 2, L-2880 in 1, 0111:B4 in 2.
MicroRNAs take part in the posttranscriptional regulation of gene expression. LPS stimulation increases expression of miR-146a (the miRNA precursor) in gingiva fibroblasts, periodontal ligament fibroblasts and dental pulp cells, upregulating KBTBD7 (gene encoding a transcriptional activator) and downregulating miR-21 and miR-155 [46,47].

Odontoblasts
E. coli LPS was used in four out of eight experiments with odontoblast-like cells. Serotypes were specified in three articles-E. coli 055:B5, E. coli k-235 strain LPS and E. coli L-2880.
Two receptors-TLR4 and CD14-bind LPS on the surface of odontoblasts [49]. LPS stimulation results in IL-8 mRNA and protein expression [50]. Binding to TLR4 upregulates VEGF (regulator of angiogenesis) expression in odontoblasts [49]. It can be associated either with regeneration of the pulp-dentinal complex or with necrosis caused by vessel collapse [49]. Odontoblasts are involved in alleviating inflammation in surrounding tissues. In response to LPS, they produce secretory leukocyte protease inhibitor (SLPI), which inhibits the activation of NF-kB and antagonizes LPS response [51]. Both mechanical injury and LPS stimulation are associated with similar Notch signaling pathway activation, which prompts differentiation of precursor cells to odontoblast-like cells [52]. This mechanism protects against injury, prevents apoptosis, enhances cell survival and maturation [52]. LPS induces inflammatory cytokines and chemokines in odontoblast-like cells, macrophages and osteoclasts. Propolis decreases production of IL-1a, MIP-1a, IL-12(p70) and IL-15 in odontoblast-like cells; MIP-1α, G-CSF, TNF-α and IL-6 in macrophages and MIP-1α production in osteoclasts [53].

DPSCs
E. coli LPS was used in 12 out of 25 experiments performed using dental pulp stem cells (DPSCs). Serotypes were specified in 6 articles-E. coli 055:B5 in two, L-2880 in one, 0111:B4 in three articles. Bindal et al. (2018) concluded that treating hDPSCs cells with 1 µg/mL E. coli LPS for 24 h is the most appropriate approach to inducing an inflammatory microenvironment, based on IL-6, IL-8, tPA and TAC1 levels [55]. LPS alone did not promote production of IL-6 in the initial phase (after 48 h), but after 7 days, rising LPS concentrations significantly increased secretion of Il-6 [56]. IL-1α level was increasing proportionally to LPS concentrations, suggesting a progressive onset of inflammation [55]. Uncontrolled overexpression of IL-1β may be the primary force driving the inflammation's severity and causing pulp necrosis [57]. Interestingly, the level of TNFα was significantly increased despite a lack of genetic expression. This phenomenon could be explained by inflammation initiation by E. coli LPS via a dual pathway-TLR4 and TLR2 [55].
Innate immune response, especially mediated by macrophages, is modulated in DP-SCs through the TNF-α/IDO axis [57]. LPS-induced TLR4 activation leads to NF-κB p65 translocation into the nucleus, with subsequent expression of iNOS, COX-2 and inflammatory mediators, such as TNF-α. The expression of the NF-κB subunit p65 is partially blocked by DPSCs and reduces TNF-α production by macrophages. IDO expression levels in DPSCs are increased after LPS or TNF-α stimulation in a time-dependent manner [57]. These mechanisms may be associated with an anti-inflammatory effect of DPSCs.

Differentiation
Wnt5a induces mesenchymal stem cells osteogenesis and suppresses adipogenesis; plays a role in tooth development and odontoblast differentiation [58]. It also upregulates inflammatory cytokines e.g., IL-6, IL-8 and IL-1b activating the non-canonical pathway [58]. LPS can enhance Wnt5a expression in a time and dose-dependent manner in hDPSCs-an effect mediated by TLR4/MyD88, PI3K/AKT and NF-kB signaling pathways [58]. LPS promotes mRNA expression of genes related to mineralization, such as OCN, DSPP, ALP and BSP in DPSCs obtained from rats and this effect declines with age [59]. LPS inhibits osteogenic differentiation of DPSCs via HMGA2/PI3K/Akt pathway [60]. He et al. (2014) [61] demonstrated that odontogenic differentiation was associated with TLR4 activation by LPS in hDPSCs. They also proved that activation of p38 mitogenactivated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK), but not NF-κB, signaling pathways are involved in LPS-mediated differentiation of hDPSCs [61]. LPS alone promotes the migration of DPSCs, however 4-Methylumbelliferone (4-mu) can further accelerate the migration and odontogenetic differentiation by downregulating the expression of inflammatory cytokines [62].
Another mechanism involved in cellular senescence is DNA damage. Excess ROS cause oxidative DNA damage (increased γ-H2A.X expression-a sensitive marker for DNA damage [64,66]), also directly activating p16INK4A expression, stopping the cell cycle [64]. LPS induces double strand breaks and subsequent DNA damage [66]. Two main classic DNA repair pathways in eukaryotic cells are homologous recombination (HR) and non-homologous end joining (NHEJ). The mRNA and protein expression levels of Ku70 and Xrcc4 involved in NHEJ, and Rad51 in HR, are increased in LPS-treated DPSCs [66]. Inhibiting Ku70 promotes the p53 pathway apoptosis [66].
LPS has a diverse effect on cell viability, defined as active cell metabolism. Some authors suggest it reduces cell viability [55], while others postulate it has no such effect [56]. Nonetheless, LPS treatment decreases cell survival by increasing apoptosis and necrosis rates [56]. LPS promotes proliferation of DPSCs by increasing TLR4 expression and through HMGA2/PI3K/Akt pathway [59,60].
As mentioned before, simvastatin presents anti-inflammatory features in hDPCs. It alleviates inflammation by downregulating TNF-α, IL-1β and MMP-9 [69]. It also increases the production of PPARγ, which is inhibited by LPS. Constructs of nanofibrous poly-(L-lactic acid) scaffolds with simvastatin trigger expression of proteins characteristic for odontoblastic differentiation [69]. This effect is mediated via phosphorylation of ERK1/2 and Smad1 [69]. LPS may influence global gene expression not only via TLRs activation, but also by changing the DNA methylation profile. P. gingivalis LPS downregulates mRNA level of DNA methyltransferase 3B (DNMT3B) and both mRNA and protein levels of DNA methyltransferase 1 (DNMT1) [70]. DNMTs inhibitor-5-Aza-CdR was used in these studies. Deregulation of above-mentioned particles increases levels of proinflammatory mediators and changes microRNAs expression [70]. The most significant change is the upregulation of miR-146a-5p, associated with pulp inflammation [71]. DNMT1 knockdown results in a similar methylation pattern of MIR146A promoter as obtained using 5-Aza-CdR [70]. LPS also induces the expression of methyltransferase-like 3 (METTL3) and extends the level of methylation at the N6 position of adenosine(m6A) [72]. Silencing METTL3 decreases the level of proinflammatory cytokines and inhibits NF-κB pathway. m6A and METTL3 regulate splicing of mRNA. Depletion of METLL3 causes production of MyD88 splice variant, which interrupts the TLR pathway [72].

Fibroblasts
Two studies examined how P. gingivalis LPS affects pulp fibroblasts. Only IL-8 (among IL-1b, IL-6, IL-8 and TNF-α measured) was detectable in LPS-stimulated fibroblasts, secreted in a time-and dose-dependent manner [83]. MicroRNA-181 family controls inflammation by regulating growth, development and activation of cells. In LPS-induced fibroblasts, miR-181a expression decreases in a time-and dose-dependent manner, miR-181b expression is barely detectable and miR-181c is absent [83]. LPS enhances chemokine (C-C motif) ligand 3 (CCL3) production in fibroblasts from permanent and deciduous teeth, whereas C-X-C motif chemokine 12 (CXCL12) production is elevated only in fibroblasts from deciduous teeth [84]. CCL3 and CXCL12 take part in leukocytes' recruitment and activation in acute inflammation.

DPSCs
LPS activates NF-κB signaling pathway in dental pulp stem cells, but its binding activities disappear by 60 min [85].

hDPCs
Nakane et al. [86] postulated that there are differences between E. coli's LPS and LPS produced by P. gingivalis, P. endodontalis and F. nucleatum. The one by E. coli inhibits cell protein production more potently than other types. All types of LPS induce DNA production by dental pulp cells [86]. Different types of LPS exacerbate the inflammation, inducing expression of pro-inflammatory cytokines. IL-1β production was enhanced by P. endodontalis LPS in cultured human gingival fibroblasts and monocytes from patients with periodontitis in a time-and dose-dependent manner [87] and by F. nucleatum LPS in a dose-dependent manner [88]. IL-1ra inhibits IL-1b synthesis induced by F. nucleatum LPS [88]. P. intermedia LPS induces IL-6 expression (probably mainly transcriptional activation) in human dental pulp fibroblast cultures time-and dose-dependently [89]. Such an IL-6 overexpression is higher than upon Salmonella LPS stimulation [89]. CD14 co-stimulates IL-6 expression in dental-pulp fibroblasts [89]. LPS upregulates the expression of both substance P (SP) and SP-receptor in DPCs, indirectly inducing expression of proinflammatory cytokines [90]. P. intermedia LPS enhances the production of VEGF in DPCs via an sCD14-dependent pathway [91].

Fibroblasts
P. intermedia (ATC 25611) LPS enhances the expression of IL-8 mRNA and its release in human dental pulp fibroblasts with a peak at 12 h [92].

Odontoblasts
P. intermedia LPS induces expression of the receptor for advanced glycation end products (RAGE) in odontoblast-like mouse cells [93]. It is a multiligand receptor propagating dysfunction of cells in several inflammatory disorders. LPS also prompts the translocation of high mobility group box 1 (HMGB1) from nucleus to secretory lysosomes, mediated by RAGE and NF-κB activation [93]. HMGB1 stimulation leads to increased expression of RAGE by human microvascular endothelial cells, production of cell adhesion molecules (ICAM and VCAM) and secretion of proinflammatory cytokines: TNFα and IL-8 [93].
Aggregatibacter actinomycetemcomitans (ATCC29524) LPS was used in one research on rat clonal dental pulp cells with odontoblastic properties [94]. Ozonated water (O3aq) suppresses calcification and immunologic responses. It also protects against direct damage to the cellular wall [94]. These effects may be achieved by directly inhibiting lipid A by O3aq [94].

DPSCs
Pseudomonas aeruginosa LPS has a toxic effect on DPSCs in a dose-dependent manner [95]. Pretreatment with static magnetic field (SMF) attenuates inflammatory response and increases viability of cells. SMF can also enhance the proliferation of DPSCs [95].

hDPCs
Several studies did not provide LPS sources. As mentioned before, LPS causes aggravation of ROS activity in cells. These particles may damage DNA and activate inflammation. LPS stimulation elevates levels of γ-H2A.X, which is a marker of double strand DNA breaks [96]. This leads to overexpression of transcription factor GATA-4, which stimulates NF-κB pathway by nuclear translocation of p65 with subsequent expression of IL-1β, IL-6 and TNF-α [96]. LPS-stimulated cells show upregulated Lin28 (RNA-binding particle) protein expression, followed by downregulation of let-7b, let-7g and miR98, and upregulation of let-7a, let-7c, let-7d, let-7e, let-7f and let-7i [97]. Let-7 family is a group of microRNAs regulating cell cycle [98].
LPS also stimulates NLRP6 expression and its knock-down decreases IL-1β production [99]. Lipopolysaccharide also upregulates both C5a and C5aR (parts of the complement system) mRNA especially on the second day post-stimulation [100]. Inflammation also raises MMP3 levels. MMP1 production increases upon TNF-α stimulation, but not LPS stimulus [101].
hDPCs possess systems counteracting excess inflammation, such as Angiogenic factor with G patch and FHA domains 1(AGGF1) [102]. It is upregulated in LPS-treated cells and its knock-down activates NF-κB via promotion of phosphorylation of p65 and its transfer to the nucleus [102]. Another similarly functioning compound is GPR173 protein [103]. It is a cognate receptor of a newly discovered hormone, phoenixin-20 [104]. GPR173 is downregulated upon LPS stimulation, leading to induced MMP-2 and MMP-9 expression [103]. However, stimulation with phoenixin-20 reverses this negative effect and decreases expression of TLR4 and MyD88 proteins [103].

Fibroblasts
LPS treatment induces labeling of membrane attack complex on the cell surface [114]. It also triggers increased production of C5a-a chemotactic factor, taking part in recruiting inflammatory cells and pulp cells responsible for regeneration [114]. LPS-stimulated fibroblasts have increased mRNA and protein expressions of myocyte-enhancer factor 2 (MEF2C), platelet endothelial cell adhesion molecule-1 (PECAM1) and CXCR4 [115]. PECAM1 expression is positively correlated with B-cell signaling pathways, it also plays a role in angiogenesis [115]. Binding PECAM1 to CXCR4 may intensify inflammation and apoptosis via NF-kB signaling pathway [115]. LPS upregulates NLRP3 inflammasome and pro-IL-1β expression via TLR4/MyD88/NF-κB pathway [116]. miR-223 is involved in the maintenance of the ATP+LPS-induced production and secretion of the proinflammatory cytokines IL-1β and IL-18 mediated by the NLRP3/CASP1 pathway, targeting NLRP3 [116].

Odontoblasts
In odontoblasts, LPS activates the transcription factor FoxO3a 24 h after LPS stimulation [117]. It is accompanied by a rise in autophagy markers, thus protecting cells from death at an early stage of inflammation [117]. The dentin-odontoblast complex may protect cells from apoptosis [118]. LPS increases production of extracellular vesicles (EVs), especially the exosomal components, attenuating apoptosis [118].
TRL4 and NOD2 are expressed in odontoblasts layer more abundantly than in other human pulp stroma cells, which can provide significant defense and anti-infection responses of the dental pulp. After stimulation of preodontoblast mouse cells with LPS, levels of TLR4, NOD2 IL-1β and autophagy proteins (LC3II, beclin1) increased. LPS induced autophagy is associated with TRL4 activation [119].

E. coli and P. gingivalis
Tenegliptin reduces production of 4-HNE from lipid peroxidation as the result of ROS activity in LPS stimulated cells. In addition, suppresses TLR4 mRNA and protein levels after LPS treatment. [26] hDPCs from healthy molar tooth unspecified Upregulated C5a and C5aR mRNA and protein expression [100] hDPCs from healthy premolar tooth E. coli 0111:B4 Upregulated lncMEG3; its knock-down inhibits the secretion of IL-1β, IL-6, TNF-α and promotes odontogenic differentiation through Wnt/β-catenin pathway. [12] hDPCs from healthy molar tooth unspecified Specific miR-146a/PEG-PEI nanoparticles combined with alginate hydrogel with basic fibroblast growth factor stimulate cell proliferation and expression of DMP-1 and DSP protein.

Cells LPS Effect in LPS-Stimulated Cells Author
hDPCs from healthy molar tooth P. gingivalis Elevated cell apoptosis (higher caspase 3 activity) inhibited cell proliferation and survival, lower SIRT6 expression. SIRT6 has anti-apoptotic effect via regulating Ku70 protein deacetylation. [74] hDPCs from healthy premolar and molar teeth

E. coli
Upregulated secretion of IL-6 and IL-8. 5-Aza-CdR can promote LPS-induced inflammation by upregulating proinflammatory cytokines expression and activating NF-κB and MAPK signaling pathways by decreasing the methylation level of TRAF6 promoter. [11] Primary human dental pulp cells were purchased from American Type Culture Collection (ATCC).

Cells LPS Effect in LPS-Stimulated Cells Author
Human pulp fibroblasts unspecified Increased mRNA and protein expression of MEF2C, PECAM1 and CXCR4. [115] Human pulp fibroblasts P. intermedia ATCC 25611 P. intermedia LPS expressed IL-8 mRNA and released IL-8 in human dental pulp fibroblast cultures, expression peaked after 12 h. [92] Human pulp fibroblasts E. coli Increased expression of miR-146a in dental pulp cells (also in gingiva and periodontal ligament fibroblasts). Decreased expression of miR-155 in gingival fibroblasts. [46] Human pulp fibroblasts from permanent and deciduous teeth

E. coli
Induced expression of IL-6, IL-8, tPA and TAC1 levels. Levels of IL-1α were increasing proportionally to LPS concentrations. TNFα showed increased expression with no changes at gene expression level. This could be attributed to initiation of inflammation via TLR4 and TLR2. The viability was reduced in LPS treated cells. [55] human dental pulp stem cells P. gingivalis, P. endodontalis LPS and TNF activated the NF-κB signaling pathway. Stimulation by the latter lasted longer. TNF induced the phosphorylation and degradation of IκBα more potently than LPS. LPS did not induce phosphorylation of p65 transactivation domain, while TNF only weakly stimulated p65 phosphorylation. [85] human dental pulp stem cells from third mandibular molars [62] human dental pulp stem cells unspecified Altered content of dental pulp cells-derived small extracellular vesicles (sEVs). sEVs carry biologically active molecules from parental cells, which can mediate intercellular communication, induce MSC differentiation, and ultimately promote the healing. [125]

LPS Effect in LPS-Stimulated Cells Author
human dental pulp stem cells from third molars E. coli 0111:B4 More pronounced SA-b-gal-positive signal (a maker of senescence), likely resulting from TLR4/MyD88-NF-jB-p53/p21 signaling pathway activation. Knockdown of p65 reversed the senescence of enhanced proliferation and the increased the total number of DPSCs with more organized F-actin. [65] human dental pulp stem cells from third molars E. coli 0111:B4 LPS binding with TLR4 generates ROS. The DDR and p16INK4A pathways might be the main mediators of DPSC LPS-induced senescence. ROS production may promote DDR and p16INK4A expression and then cell cycle arrest. [64] human dental pulp stem cells from premolars or third molars E. coli 055:B5 SDF-1a secretion largely suppressed, increased production of CXCR4. [63] human dental pulp stem cells from third molars Ultrapure E. coli LPS can enhance Wnt5a expression preincubation with TLR4 neutralizing antibodies reduced LPS-induction Wnt5a expression (mainly activated through the MyD88-dependent pathway). NF-kB activation and PI3K/AKT signal pathways regulate Wnt5a expression. [58] human dental pulp stem cells Ultrapure E. coli Induced odontogenic differentiation by increasing ALP, OCN, DMP1 and DSPP expression and mineralized nodules formation. TLR4 expression maximal after 7 days. Inhibiting or blocking TLR4 decreased the LPS-mediated expression of mineralized tissue markers and nodule formation. NF-kB signaling activated by LPS in a time-dependent manner, but not involved in cellular differentiation. Activation of p38 and ERK MAPK, not JNK MAPK signaling pathways contribute to LPS-induced differentiation of hDPSCs. [61] human dental pulp stem cells from wisdom teeth/premolars P. aeruginosa Dose-dependent toxic effect. (SMF) stimulation inhibits inflammatory response, can enhance proliferation. [95] human dental pulp stem cells from third molars unspecified Exosomes from LPS-stimulated hDPSCs exert a stronger pro-angiogenic effect on HUVECs than normal hDPSCs-LPS-dose dependently. Expression of 7 microRNAs were increased (miR-146a-5p, miR-92b-5p, miR-218-5p, miR-23b-5p, miR-2110, miR-27a-5p, and miR-200b-3p) and 3 decreased (miR-223-3p, miR-1246 and miR-494-3p). Five of them play important roles in inflammation and HUVEC function and angiogenesis (miR-223-3p being the strongest candidate). [121] human dental pulp stem cells from premolars E. coli 0111:B4 High expression of γ-H2A.X. marker of DSB. The mRNA and protein expression levels of Ku70 and Xrcc4 involved in NHEJ, and Rad51 in HR, significantly increased in DPSCs. Ku70 knockdown reduces the expression of XRCC4 and promotes apoptosis of DPSCs during inflammation, thereby Ku70 serves as a link between DNA damage and apoptosis. [66] human dental pulp stem cells from third molars E. coli Upregulated CD146 expression levels. Partially blocked expression of the NF-κB subunit p65 leading to reduced TNF-α production by macrophages. The innate immune response dependent on the TNF-α/IDO axis. [57] human dental pulp stem cells from third molars E. coli EGCG exerted an anti-inflammatory effect on hDPSCs without affecting cell proliferation or differentiation. EGCG inhibits hypoxia-induced apoptosis. [67]

LPS Effect in LPS-Stimulated Cells Author
rat dental pulp stem cells E. coli 055:B5 LPS at low concentrations upregulated mRNA expression of mineralization-related genes (OCN, DSPP, ALP and BSP) in JDPSCs and ADPSCs, LPS effects declined with age. Enhanced proliferation by increasing TLR4 expression and through PI3K/Akt signaling. [59] human dental pulp stem cells from third molars unspecified VEGFA promoted the migration of hDPSCs in a concentration-dependent manner. VEGFA/VEGFR2 axis interacted with the FAK/PI3K/Akt and p38 MAPK signaling pathways in mediating hDPSCs migration. [126] human dental pulp stem cells from premolars (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED)

E. coli
Betamethasone blocks NF-κB activation and exhibits an osteo-/odonto-inductive effect on DPSCs and SHED. It also displays an osteoclast effect on SHED, not on the DPSCs. [68] rat dental pulp stem cells unspecified The expression of miR-506 was high while that of SIRT1 was low, which was associated with pro-inflammatory cytokines upregulation and activation of the TLR4-NFkB pathway. [122] human dental pulp stem cells from third molars E. coli O55:B5 Decreased cell survival and more frequent necrosis. Reduced COL1A1 expression after 21 d. Promoted production of IL-6 in the late phase. [56] human dental pulp stem cells from third molars unspecified Simvastatin promoted cell proliferation, cell cycling and apoptosis in LPS-induced DPSCs. Expression of cytokines and VEGF (via MAPK signaling blockade) was inhibited. [128] rat dental pulp stem cells unspecified Inhibited expression of let-7c-5p both in vivo and in vitro. The overexpression of let-7c-5p suppressed the production of pro-inflammatory cytokines, restoring viability; also inhibited the LPS-induced activation of NF-kB signaling by inhibiting the phosphorylation of IκBa and IKKb and increasing total IκBa expression, hence suppressing the nuclear translocation of NF-kB p65. let-7c-5p action depends on the inhibition of DMP1 function. [120] rat dental pulp stem cells E. coli 055:B5 Induced expression of let-7c-5p could suppress inflammation and restored the osteogenic differentiation potential of inflamed DPSCs. The effect depended on the repression on HMGA2 function by let-7c-5p, leading to inhibiting PI3K/Akt pathway. [60] human dental pulp stem cells unspecified Human β-Defensin 4 (HBD4) shows anti-inflammatory activity in vitro, by reduction of IL-1α, IL-1β, IL-6 and TNF-α expression and promotes mineralizing cell phenotype differentiation in DPSC. Similar effects are noted in vivo. [123] human dental pulp stem cells-normal pulp derived from the mandibular third molar and inflamed pulps derived from pulps of patients with irreversible pulpitis unspecified Decreased Wnt4 expression, impairing the odontogenic differentiation of DPSCs. Restoration of Wnt4 was able to rehabilitate the impaired odontogenic differentiation potential.

E. coli
Upregulated VEGF expression (TLR4-dependent signaling pathway). Therapeutic blockade of the LPS-TLR4-VEGF pathway might be beneficial for the treatment of teeth with reversible pulpitis.
[49] Odontoblast-like cells produce secretory leukocyte protease inhibitor (SLPI) in response to LPS, inhibiting the activation of NF-kB. [51] Human odontoblasts from third molars E. coli 055:B5 Upregulated IL-8 mRNA and protein levels. [50] self-established pre-odontoblastic cell line from third molars unspecified During the early stage of inflammation, FoxO3a might regulate autophagy activation for odontoblast survival. [117] Mouse odontoblast-like cells E. coli L-2880 Notch signaling activation by LPS stimulation is similar to that caused by mechanical injury in vivo. [52] rat clonal dental pulp cell line with odontoblastic properties (KN-3) A. actinomycetemcomitans ATCC29524 O3aq directly suppresses the biological effects of LPS on calcification and immunologic responses of odontoblast-like cells and its ability to demolish cell walls and cytoplasmic membranes.
O3aq effects may be achieved through the direct inhibition of lipid A. [94] Human Dental Pulp Tissues; Odontoblast-like cells, OLC-1, obtained from mouse tooth germs
LPS changes the content of small extracellular vesicles (sEVs), promoting dental pulp regeneration by increasing stem cells migration and elevating the expression levels of repair-associated proteins [125]. Exomes isolated from LPS-induced HDPSCs present a stronger pro-angiogenic effect on human umbilical vein endothelial cells (HUVECs) than HDPSCs without LPS stimulation [121]. A time-and dose-dependent increase in VEGFA expression is also noted [126]. In VEGFA-stimulated hDPSCs, FAK, PI3K, Akt and p38 signaling are activated, possibly enhancing cellular migration [126].
Human platelet lysate (HPL) is a growth factors-rich concentrate of platelets. 20% HPL increases expression of pro-angiogenic factors at both gene and protein levels, while maintaining the cell viability [127]. In LPS-induced DPSCs simvastatin inhibits the expression of proinflammatory cytokines and VEGF, blocking the MAPK signaling [128]. It also promotes proliferation and apoptosis [128].

Discussion
In this study, we summarize in vitro research concerning LPS influence on different dental pulp cells. Results show that bacterial endotoxins activate a variety of cellular pathways. Figures 2-5 give an overview of such mechanisms in particular types of cells. We combined data extracted from analyzed studies with information obtained from KEGG database [129]. Despite some structural differences in LPS originating from miscellaneous bacteria species, they are recognized by only two types of TLR receptors-TLR4 and TLR2, which act via the same pathway. Hence, we simplified the graphical presentation of data, not differentiating between various LPS origins. The main response axis in dental cells begins with activation of TLR4/MyD88/NF-κB pathway, with co-activation of MAP kinases. These pathways are responsible for increasing expression of interleukins, chemokines, MMPs, TNF-α and adhesion molecules. LPS stimulation alters cellular functions in complex ways. Firstly, acting through TLR2/4 receptors, it activates production of inflammatory compounds. Subsequently, gene expression is altered by DNA methylation or expression of microRNAs. LPS also induces autophagy. Finally, LPS stimulation may result in apoptotic or pyroptotic death of affected cells.
An indirect effect of LPS on the inflammation apparatus is exerted by raising ROS production. These factors induce both MAPK and NF-κB pathways, but also inflict DNA damage. This may induce repairing systems or prompt apoptosis, if uncontrolled. Interestingly, agonistic stimulation of PPARγ, which is downregulated in LPS-treated cells, can decrease ROS activity [17].
Apart from TLRs, the NLRP system is also involved in signal transduction after exposure to LPS. In selected studies two NLRPs were described-NLRP3 and NLRP6. They both are involved in transducing the signal for Casp1, resulting in pyroptotic cell death.
One of the well-described pathways in hDPCs and odontoblasts is HMGB1 pathway. In LPS-treated cells, the level of HMGB1 is increased. A potential regulator of this protein is microRNA let-7c-5p, which is downregulated by NUTM2A-a particle expressed abundantly after LPS stimulation. HMGB1 through activation of RAGE receptors deteriorates cellular viability and suppresses proliferation.
The analyzed studies present ambiguous results regarding odontoblastic differentiation. On the one hand, authors show results proving that LPS stimulates cells to become odontoblasts [39,61]-an effect regulated by increased levels of DMP-1, DSPP and ALP activity. An alternative pathway mediating this process may be through activation of Wnt5 or AP-1 and MAPK [40]. On the other hand, however, there are several studies with opposing results. Inhibition of Wnt4 pathway or higher expression of dentine-related proteins occurred in cells treated with compounds reducing expression of proinflammatory interleukins [28,82,124].   The conclusions of the studies presented above should be interpreted with caution, taking into consideration potential biases. Firstly, there is some uncertainty about cells called hDPCs, which were used in most selected studies. As mentioned in the introduction, dental pulp contains a variety of cell types. In most studies, cell isolation methods did not ascertain the purity of isolated cell lineage. Likely, hDPCs are in fact cohorts of nonhomogenous cells present in the dental pulp. This poses a risk of bias when comparing study results.
with their names. Black arrows represent positive stimulation, dashed arrows show indirect activation, red ones-inhibition, yellow-u translocation to another cell compartment. Created with BioRender.com 7.12.2020.   Figure summarizes results from studies conducted on DPSCs, regardless of LPS origin. The main pathway is TLR/MyD88/NF-κB. This pathway is marked with thick blue arrows. Its activation increases interleukins production and odontoblastic differentiation. The last one is also mediated by Wnt4/5 pathways. LPS stimulation also causes expression of membranous CD44 and CD146. Variety of different pathways and particles activated in LPS-treated cells are presented in both deep blue brackets and by protein images with their names. Black arrows represent positive stimulation, dashed arrows show indirect activation, red ones-inhibition, green-translocation to another cell compartment. Created with BioRender.com 7 December 2020.
Inflammation is a stressful condition, which changes cellular metabolism. It activates a variety of proteins; therefore, it is also important to consider potential bias associated with crosstalk between different pathways. Most studies analyzed only one particle and potential downstream transcriptional molecules. Such an approach does not consider the potential impact of other molecular mechanisms that may have a reciprocal effect on each other. For example, in studies investigating potential anti-inflammatory compounds, a given molecule may activate some transduction pathways, even though it attenuates the inflammation. To ensure the quality of future studies, proper defining of the phenotype of utilized cells should be a standard practice.
arrows represent positive stimulation, dashed arrows show indirect activation, red ones-inhibition, green-translocation to anot with BioRender.com 7.12.2020.

Conclusions
In summary, LPS affects various proinflammatory pathways in pulp cells. The main role is played by TLR receptor activation followed by NF-κB stimulation. Other important molecules in LPS-stimulated inflammation are NLRs and ROS. Changes in cells treated with LPS are exerted at all levels of expression regulation, from DNA methylation to mRNA post-translational modification.