Increased Innate Lymphoid Cells in Periodontal Tissue of the Murine Model of Periodontitis: The Role of AMP-Activated Protein Kinase and Relevance for the Human Condition

Abstract

Innate lymphoid cells (ILCs) are master regulators of immune and inflammatory responses, but their own regulatory mechanisms and functional roles of their subtypes (i.e., ILC1s-ILC3s) remain largely unresolved. Interestingly, AMP-activated protein kinase (AMPK), influences inflammatory responses, but its role in modulation of ILCs is not known. Periodontitis is a prevalent disorder with impairment of immune and inflammatory responses contributing importantly to its pathogenesis; however, neither the role of ILCs nor AMPK has been explored in this condition. We tested the hypotheses that (a) periodontitis increases ILCs and expression of relevant cytokines thereby contributing to inflammation and (b) knockdown of AMPK worsens indices of periodontitis in association with further increases in subtypes of ILCs and cytokine expression. The studies utilized wild-type (WT) and AMPK knockout (KO) mice, subjected to ligature-induced periodontitis or sham operation, in association with the use of micro-CT for assessment of bone loss, immunogold electron microscopy to show presence of ILCs in periodontal tissues, flow cytometry for quantitative assessment of subtypes of ILCs and RT-polymerase chain reaction analyses to measure mRNA expression of several relevant cytokines. The results for the first time show (a) presence of each subtype of ILCs in periodontal tissues of sham control and periodontitis animals, (b) that periodontitis is associated with increased frequencies of ILC1s-ILC3s with the effect more marked for ILC2s and differential phenotypic marker expression for ILC3s, (c) that AMPK KO mice display exacerbation of indices of periodontitis in association with further increases in the frequency of subtypes of ILCs with persistence of ILC2s effect, and (d) that periodontitis increased mRNA for interleukin (IL)-33, but not IL-5 or IL-13, in WT mice but expression of these cytokines was markedly increased in AMPK KO mice with periodontitis. Subsequently, we showed that human periodontitis is associated with increases in each ILCs subtype with the effect more marked for ILC2s and that mRNA expressions for IL-33 and IL-5 are markedly greater for sites affected by periodontitis than healthy sites. Collectively, these novel observations indicate a pivotal role for ILCs in pathogenesis of periodontitis and that AMPK is a regulator of their phenotype expression in this condition.

Keywords: AMP-activated protein kinase; cytokines; human; inflammation; innate lymphoid cells; periodontitis.

Figure 1

Ligature-induced periodontitis model in mice and presence of innate lymphoid cells (ILCs) in periodontal tissues. (A) Gross view of ligature-induced periodontitis, second molar, after 7 days of suture placement; arrows point to the suture being covered by palatal tissue and consequent bone loss around the ligated tooth; scale bar = 1 mm. (B) Sagittal, transverse, and 3-D micro-CT images of second molar and associated structures. Micro-CT images show marked bone loss in response to placement of suture compared to sham operation. Bar graphs show alveolar bone loss fraction around mesial and distal roots (C) and bone volume fraction on right and left side second-molar teeth (D) in experimental groups. (E) Transmission electron microscopy images of periodontal tissues obtained from sham-operated control and ligature-induced periodontitis mice. Each panel shows one cell displaying two-sized gold particles; large particles (red arrows) confirming specific markers (intracellular and nuclear/perinuclear stains) identifying ILC1s, ILC2s, and ILC3s. *p < 0.05 compared to the other condition/group.

Figure 2

Effect of AMP-activated protein kinase (AMPK) knockdown on murine periodontitis model. Alveolar bone loss fraction around mesial and distal roots of the second molar in sham-operated and ligature-induced periodontitis in wild-type (WT) mice and AMP-activated protein kinase knockout (AMPK KO) mice. The inset shows successful genetic deletion of WT α1 AMPK as revealed by the absence of polymerase chain reaction band at 450 bp while the mutant band was detected at 350 bp [inset panel (A); tail tissue] and flow cytometry for AMPK protein expression [inset panel (B); peripheral blood] thereby confirming success of AMPK deletion in AMPK KO compared to WT mice. *p < 0.05 compared to their sham-operated counterparts. ^#p < 0.05 compared to WT/periodontitis group.

Figure 3

Innate lymphoid cells frequency and subtypes. Representative panels show multiparameter flow cytometry and gating strategy to determine subsets of innate lymphoid cells (ILCs) in experimental groups as described under Section “Materials and Methods.” Briefly, ILCs were initially identified as lineage negative CD127⁺ lymphocytes followed by analyses of signature transcription factor for each subset of ILCs as follow: ILC1s: T-bet; ILC2s: GATA-3, and ILC3s: RORγt (A). (B) Average ± SEM of each ILCs’ subtype for sham-operated and ligature-induced periodontitis in wild-type (WT) and AMP-activated protein kinase knockout (AMPK KO) mice. (C) Further flow cytometry-based evaluation for expression of specific markers for each subset of ILCs as follow: ILC1s: CD127 and CD49a; ILC2s: ST2 and killer cell lectin-like receptor G1 (KLRG1); ILC3s: NKp46 and CCR6; the assessment was carried out without or with prior in vitro treatment with relevant cytokines as shown for each subset of ILCs as described under Section “Materials and Methods.” Value in each quadrant represents percent of total cells for each group/condition/treatment; ^#quadrants with most noted changes between the two groups under each condition. *p < 0.05 compared to their sham-operated counterparts.

Figure 4

Cytokine expression in periodontal tissues of mice. Bar graphs show mRNA expression for cytokine expression in periodontal tissues obtained from sham-operated and ligature-induced periodontitis in wild-type (WT) and AMP-activated protein kinase knockout (AMPK KO) mice. *p < 0.05 compared to their sham-operated counterparts. ^#p < 0.05 compared to interleukin (IL)-33 expression in the same group.

Figure 5

Innate lymphoid cells frequency and subtypes in human periodontitis. Representative panels show multiparameter flow cytometry gating strategy to determine subsets of innate lymphoid cells (ILCs) in experimental groups as described under Section “Materials and Methods.” Briefly, ILCs were initially identified as lineage negative CD127⁺ lymphocytes followed by analyses for signature molecules for each subset of ILCs as follow: ILC1: T-bet; ILC2: GATA-3 and ILC3: RORγt (A). (B) Average ± SEM of each ILCs subtype for periodontal tissues obtained from healthy sites and those affected by periodontitis of human subjects. *p < 0.05 compared to healthy control sites.

Figure 6

Cytokine expression in human periodontal tissues. Bar graphs show mRNA expression for cytokine expression in periodontal tissues obtained from healthy sites and those affected by periodontitis of human subjects. *p < 0.05 compared to same cytokine in the control group. ^#p < 0.05 compared to interleukin (IL)-33 or IL-5 expression in the same group.