Gene expression data of inflammatory mediators in apical periodontitis in 129 (wild type) and 5-lipoxygenase knockout mice.

Apical periodontitis is an immune inflammatory response around periapical tissues as a result of pathogens invasion into the root canal. The host immunoinflammatory response could determine the progression of this disease, which involves the recruitment of immune cells, and the release of several cytokines in the lesion site. The 5-lipoxygenase pathway has been activated in some osteolytic diseases due to its capacity to interfere in the proliferation and differentiation of bone cells, including the osteoclasts. As mean to understand the inflammatory genes regulation in the apical periodontitis progression, we evaluated the network of 66 genes related to cytokines, chemokines and other inflammatory mediators and receptors in the wild-type (WT) and 5-lipoxygenase enzyme genetically deficient mice (KO). This article presents data not published but related to the research article "Effects of 5-lipoxygenase gene disruption on inflammation, osteoclastogenesis and bone resorption in polymicrobial apical periodontitis" .

Specifications Table

Value of the Data

The data shows a panorama of inflammatory genes profile in the apical periodontitis progression in the wild-type and 5-lipoxygenase enzyme knockout mice.

This data provides a valuable tool for studying the apical periodontitis development by comparing the inflammatory genes expression modulation in both animals using heatmap and gene regulatory networks in both models.

The data could contribute to interpretation of 5-lipoxygenase enzyme to the inflammatory genes expression network during the apical periodontitis development.

Data Description

The 5-lipoxygenase enzyme deficiency in mice can result in changes in the immunoinflammatory markers gene expression during the apical periodontitis development. Furthermore, the absence of this enzyme affects gene interaction resulting in a broader network at 28 days of the disease. Table 1 integrates symbol and nomenclature of genes evaluated by qRT-PCR. The raw data of qRT-PCR analysis of each gene can be found in the Supplementary file. Fig. 1 shows a heatmap with 66 inflammatory mediator genes evaluated in the apical periodontitis of WT and KO at different stages of the disease, after 7, 14, 21 and 28 days of apical periodontitis. Gene regulatory network was evaluated in wild type (Fig. 2) and in knockout mice (Fig. 3), both at 28 days after apical periodontitis induction. In Figs. 2 and 3, the regulatory genes (Hub gene) and connected genes (nodes genes) of each group were shown.

Table 1

National Center for Biotechnology Information (NCBI) of the transcriptome, Bank access number, Symbol and Nomenclature of genes evaluated by qRT-PCR array that encode mouse inflammatory cytokines and receptors.

Unigene	GeneBank	Symbol	Nomenclature
Mm.329022Mm.347398Mm.491799Mm.19131Mm.1051Mm.4686Mm.867Mm.41988Mm.490604Mm.290320Mm.116739Mm.12895Mm.31505Mm.7275Mm.341574Mm.416125Mm.274927Mm.6272Mm.57050Mm.1337Mm.14302Mm.8007Mm.2932Mm.442098Mm.442383Mm.28767Mm.21013Mm.303231Mm.332490Mm.12876Mm.8021Mm.240327Mm.379327Mm.4154Mm.35814Mm.24208Mm.490053Mm.10137Mm.59313Mm.1410Mm.133095Mm.45901Mm.896Mm.1349Mm.384038Mm.2923Mm.983Mm.276360Mm.3448Mm.2856Mm.4364Mm.234466Mm.262106Mm.1137Mm.87787Mm.1715Mm.2326Mm.235137Mm.288474	NM_013854NM_009744NM_007551NM_009778NM_009807NM_011330NM_011331NM_011332NM_011888NM_011333NM_016960NM_009137NM_019577NM_009138NM_013654NM_011338NM_009912NM_009915NM_009914NM_009916NM_009917NM_009835NM_007719NM_007720NM_009913NM_007768NM_008176NM_021704NM_019932NM_009910NM_007721NM_008337NM_008348NM_008349NM_008350NM_133,990NM_008357NM_010551NM_019508NM_008360NM_019450NM_027163NM_008362NM_010555NM_008368NM_013563NM_010556NM_021283NM_008370NM_010559NM_010560NM_009909NM_008401NM_008404NM_010735NM_008518NM_010798NM_007926NM_009263	Abcf1Bcl6Cxcr5C3Casp1Ccl11Ccl12Ccl17Ccl19Ccl2Ccl20Ccl22Ccl24Ccl25Ccl7Ccl9Ccr1Ccr2Ccr3Ccr4Ccr5Ccr6Ccr7Ccr8Ccr9CrpCxcl1Cxcl12Pf4Cxcr3Ccr10IfngIl10raIl10rbIl11Il13ra1Il15Il16Il17bIl18Il1f6Il1f8Il1r1Il1r2Il2rbIl2rgIl3Il4Il5raIl6raIl6stCxcr2ItgamItgb2LtaLtbMifAimp1Spp1	ATP-binding cassette, sub-family F (GCN20), member 1B-cell leukemia/lymphoma 6Chemokine (C-X-C motif) receptor 5Complement component 3Caspase 1Chemokine (C—C motif) ligand 11Chemokine (C—C motif) ligand 12Chemokine (C—C motif) ligand 17Chemokine (C—C motif) ligand 19Chemokine (C—C motif) ligand 2Chemokine (C—C motif) ligand 20Chemokine (C—C motif) ligand 22Chemokine (C—C motif) ligand 24Chemokine (C—C motif) ligand 25Chemokine (C—C motif) ligand 7Chemokine (C—C motif) ligand 9Chemokine (C—C motif) receptor 1Chemokine (C—C motif) receptor 2Chemokine (C—C motif) receptor 3Chemokine (C—C motif) receptor 4Chemokine (C—C motif) receptor 5Chemokine (C—C motif) receptor 6Chemokine (C—C motif) receptor 7Chemokine (C—C motif) receptor 8Chemokine (C—C motif) receptor 9C-reactive protein, pentraxin-relatedChemokine (C-X-C motif) ligand 1Chemokine (C-X-C motif) ligand 12Platelet factor 4Chemokine (C-X-C motif) receptor 3Chemokine (C—C motif) receptor 10Interferon gammaInterleukin 10 receptor, alphaInterleukin 10 receptor, betaInterleukin 11Interleukin 13 receptor, alpha 1Interleukin 15Interleukin 16Interleukin 17BInterleukin 18Interleukin 1 family, member 6Interleukin 1 family, member 8Interleukin 1 receptor, type IInterleukin 1 receptor, type IIInterleukin 2 receptor, beta chainInterleukin 2 receptor, gamma chainInterleukin 3Interleukin 4Interleukin 5 receptor, alphaInterleukin 6 receptor, alphaInterleukin 6 signal transducerChemokine (C-X-C motif) receptor 2Integrin alpha MIntegrin beta 2Lymphotoxin ALymphotoxin BMacrophage migration inhibitory factorAminoacyl tRNA synthetase complex-interacting multifunctional protein 1
Mm.248380Mm.1293Mm.474976Mm.235328Mm.4861Mm.103551Mm.390241Mm.3317Mm.299381Mm.2180Mm.304088Mm.391967	NM_011577NM_013693NM_011609NM_011610NM_011616NM_023764NM_011798NM_010368NM_013556NM_008302NM_008084NM_007393	Tgfb1TnfTnfrsf1aTnfrsf1bCd40lgTollipXcr1GusbHprtHsp90ab1GapdhActb	Secreted phosphoprotein 1Transforming growth factor, beta 1Tumor necrosis factorTumor necrosis factor receptor superfamily, member 1aTumor necrosis factor receptor superfamily, member 1bCD40 ligandToll interacting proteinChemokine (C motif) receptor 1Glucuronidase, betaHypoxanthine guanine phosphoribosyl transferaseHeat shock protein 90 alpha, class B member 1Glyceraldehyde-3-phosphate dehydrogenaseActin, beta

Fig. 1

Heatmap and cluster analysis of kinetic of 66 genes expression in the apical periodontitis of WT and KO mice at 7, 14, 21 and 28 days of lesion and their respective control groups. Color codes in each panel refer to blue for low expression and red for the highest expression levels.

Fig. 2

Gene regulatory network (GRN) using the Graphical Lasso (λ = 0.300) method of WT mice at 28 days of lesion. Yellow circles indicate regulatory genes (hub gene) and light blue circles indicate poorly connected genes (nodes genes). There is 1 hub gene: CXCL10 participates in gene regulation and biological processes.

Fig. 3

Gene regulatory network (GRN) using the Graphical Lasso (λ = 0.300) method of KO mice at 28 days of lesion. Yellow circles indicate regulatory genes (Hub gene) and light blue circles indicate poorly connected genes (nodes genes). There are 5 hub genes: IL-1β, IL-3, IL-20, CXCL9 and CCL3 participate in gene regulation and biological processes.

Experimental Design, Material and Methods

Animals

Twenty four knockout (KO) mice for 5-lipoxygenase enzyme (129-Alox5tm1Fun; 129-Alox5-/-; The Jackson Laboratory, Bar Harbor, ME, USA) and 24 wild-type 129 mice for the control group were used in this study. Mice were male and adult (6–8 week-old). For the operative procedures the animals were anesthetized by intraperitoneal injections of ketamine hydrochloride (150 mg/kg, Ketamine 10%, Agener União Química Farmacêutica Nacional S/A, Embu-Guaçu, SP) and xylazine (7.5 mg/kg, Dopaser, Laboratorios Calier S/A, Barcelona, Spain).

Apical periodontitis model

The protocol of apical periodontitis was previously described in Da Silva et al. [2]. Mice were placed in a surgical table in order to promote the immobilization of the animals, maintenance of the mouth opened, and the visualization of the molar teeth. The upper first molar pulps were exposed using a 1011 spherical diamond tip (KG Sorensen Ind. Com. Ltda., Barueri, SP) and a type K file #06 (Les Fils d'Auguste Maillefer S/A, Switzerland). The exposed root canals were left open to the oral environment, as previously described [3]. The teeth without pulp exposure were used as controls. Mice were euthanized on days 7, 14, 21 and 28 after experimental apical periodontitis induction (n = 6 teeth per period).

Evaluation of gene expression by global qRT-PCR arrays to demarcation of inflammatory event

A guanidine thiocyanate protocol (RNeasy® Mini, Qiagen Inc., Valencia, USA) was used for RNA extraction from two pools of three teeth each. The evaluation of the total RNA quality was performed by electrophoresis on 1% agarose gel (Sigma-Aldrich Corp.) containing ethidium bromide (Sigma-Aldrich Corp.) using 1x concentrated TBE buffer (Tris-Borate-EDTA). The estimate of the amount of nucleic acids and their purity were assessed by spectrophotometry in NanoDrop 1000 (Thermo Fisher Scientific Inc., Wilmington, USA). The synthesis cDNA via reverse transcription reaction was performed by using 2 µg of total RNA and the First Strand RT2 kit (Qiagen Inc.).

RT-PCR arrays (Inflammatory Cytokines and Receptors PAMM-011Z, Qiagen Inc.) were used for the analysis of 66 target sequence genes (Table 1). As reference genes, Gusb, Hprt, Hsp90ab1, Actb and Gapdh were evaluated. Controls for detecting mouse genomic DNA contamination (MGDC), controls for the efficiency of the reverse transcription reaction (RTC) and the positive controls (PPC) consisting of a passive artificial DNA sequence to be detected during the reaction. The qRT-PCR reactions were performed using SybrGreen, consisting of a duplicate in an Eppendorf Mastercycler® ep Realplex (Eppendorf AG). Amplification was done under the following conditions: denaturation at 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The specificity of the primers was analyzed using the dissociation curve, considering the melting temperature of the amplicon under the following conditions: temperature increase to 95 °C for 15 s, followed by decrease the temperature to 60 °C for 15 s, gradually increasing the temperature to 95 °C for 20 min and maintained at 95 °C for 15 min. The ΔΔCt method was used for relative quantification.

Data presentation and analysis

The qRT-PCR data of 66 gene expressions were plotted in MS Excel for the data normalization. The relative quantification of all experimental groups were analyzed by R statistical package version 4.0.3. For data analysis, a heatmap was used in order to show the magnitude of the fold change of each gene in a color scale. The columns correspond to the experimental groups and the rows the genes evaluated.

Gene regulatory networks

The gene-gene association network of the same 66 genes was evaluated in the WT and KO group, both with 28 days of apical periodontitis, using the Graphical Lasso method (λ = 0.300) by GeneCK, a web server for building gene networks and visualization [4]. These graphs shows the nodes representing the genes and the edges representing the gene-gene interaction.

Ethics Statement

All experiments using animals were performed following the guidelines for animal research at University of São Paulo (USP). The experimental protocols were approved by the Ethics Committee on Animal Use from the School of Dentistry of Ribeirão Preto (process# 12.1.60.53.8).

CRediT authorship contribution statement

Thaise Mayumi Taira: Software, Data curation, Writing – review & editing. Vítor Luís Ribeiro: Software, Data curation, Writing – review & editing. Yuri Jivago Silva Ribeiro: Writing – review & editing. Raquel Assed Bezerra da Silva: Supervision, Writing – review & editing. Léa Assed Bezerra da Silva: Supervision, Writing – review & editing. Marília Pacífico Lucisano Politi: Supervision, Writing – review & editing. Lúcia Helena Faccioli: Conceptualization, Supervision. Francisco Wanderley Garcia Paula-Silva: Conceptualization, Methodology, Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare no conflict of interest for this article.

Subject	Dentistry, Oral Surgery and Medicine
Specific subject area	Endodontics
Type of data	Gene expression (fold change) relative to control
How data were acquired	RNA extraction followed by reverse transcriptionAmplification in qRT-PCR machine (40 cycles)Instruments: Step one Plus (Applied Biosystems), GeNeCK web server and Software R
Data format	RawGraphsFiguresTable in Excel datasheetReport relative expressionAnalyzed
Parameters for data collection	Jaw samples with apical periodontitis and control (contralateral jaw with healthy teeth without lesion) from 5-lipoxygenase enzyme knockout mice and wild-type mice were collected as described [1,2]. Several inflammatory mediators were evaluated by qRT-PCR [1].
Description of data collection	Inflammatory mediators gene expression array was compared at different time points of apical periodontitis in the 5-lipoxygenase enzyme knockout mice and compared to wild-type mice.
Data source location	Institution: Universidade de São PauloCity/Town/Region: Ribeirão PretoCountry: Brazil
Data accessibility	Full data is host in a public repository. Repository name: Universidade de São Paulo. Direct URL to data:http://repositorio.uspdigital.usp.br/handle/item/336
Related research article	Paula-Silva, F., Arnez, M., Petean, I., Almeida-Junior, L. A., da Silva, R., da Silva, L., & Faccioli, L. H. (2020). Effects of 5-lipoxygenase gene disruption on inflammation, osteoclastogenesis and bone resorption in polymicrobial apical periodontitis. Archives of oral biology, 112, 104,670. 10.1016/j.archoralbio.2020.104670