Macrophage M1 polarization mediated via the IL-6/STAT3 pathway contributes to apical periodontitis induced by Porphyromonas gingivalis

Abstract Objective: To investigate the involvement of IL-6/STAT3 signaling pathway activation in macrophage polarization and bone destruction related to apical periodontitis (AP) stimulated by Porphyromonas gingivalis. Methodology: Macrophage polarization, IL-6/STAT3 expression, and the presence of P. gingivalis were detected in human AP tissues via RT-qPCR, western blotting, and immunohistochemistry staining. Murine bone marrow derived macrophages were isolated and cultured with P. gingivalis W83 in vitro, and levels of macrophage IL-6 expression, STAT3 phosphorylation, and macrophage polarization with or without the selective STAT3 phosphorylation inhibitor Stattic (5 μM) were detected via ELISA, western blotting, RT-qPCR, and flow cytometry, respectively. P. gingivalis-induced murine AP models were constructed, and bone destruction and macrophage polarization in the apical region were evaluated. Transwell co-culture systems were used to investigate the effects of macrophages infected with P. gingivalis on osteogenesis and osteoclastogenesis. Results: P. gingivalis was detected in human AP tissues that highly expressed IL-6/STAT3, and the M1 subtype of macrophages was more abundant in these tissues. P. gingivalis infection induced IL-6 expression, STAT3 phosphorylation, and M1 polarization of macrophages, while 5 μM of Stattic partially abolished these activation effects. Systemic STAT3 blockade via oral administration of Stattic at a dose of 25 mg kg-1 alleviated murine periapical bone resorption and apical infiltration of M1 macrophages induced by P. gingivalis infection in vivo. Furthermore, macrophages infected with P. gingivalis promoted bone destruction via secretion of IL-6, TNF-α, and RANKL, which hinder pre-osteoblast expression of Runx2 and accelerate pre-osteoclast expression of NFAT2. Conclusions: The activation of IL-6/STAT3 signaling pathway is involved in mediating macrophages M1 polarization in the P. gingivalis induced apical inflammatory context and may also be intimately involved in the bone loss caused by P. gingivalis infection, directing the M1 macrophage infiltration during the progression of AP.


Introduction
Apical periodontitis (AP) is an infectious inflammatory lytic bone lesion, primarily caused by microbial infection of the root canal system. 1,2 Invasion of bacteria and their components elicits a nonspecific host response in periapical tissues that results in local inflammation, infiltration of immune cells, resorption of hard tissues, formation of granulation tissue, and eventual development of various periapical lesions. 3,4 Porphyromonas gingivalis, the so-called keystone pathogen in periodontitis, 5 is detected not only in the biofilm in periodontal pockets, but also as a major bacterial species in infected pulp chambers and root canals with AP. 3,6,7 However, the mechanism underlying the involvement of P. gingivalis in the pathology of AP has yet to be elucidated. P. gingivalis uses several strategies to interact with the host defense system, escape host immune surveillance, and initiate the inflammation process; this bacterial pathogen can invade and survive inside endothelial cells, osteoblasts, and macrophages. 8-10 By activating Toll-like receptors (TLRs), P. gingivalis induces the expression of cytokines including interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and lymphotactin in murine macrophages, 11,12 causing murine bone loss. [13][14][15] Macrophages are indispensable immunocompetent infiltrates in the development of AP. 16 M1 and M2 polarized macrophages are two major types of activated macrophages. 17 Classically activated M1 macrophages produce pro-inflammatory and bone-destructive mediators. such as IL-1α, IL-6, IL-1β, and tumor necrotic factor-α (TNF-α), which lead to cytotoxicity, tissue injury, and excessive osteoclastic activity. In contrast, alternatively activated M2 macrophages secrete anti-inflammatory mediators, such as IL-10 and arginase 1, which regulate the level of chronic inflammation and are further associated with tissue homeostasis, resolution of inflammation, and tissue repair. 16,[18][19][20] Recently, however, the immunomodulatory state of M2 macrophages has been found to be related to maintenance of periapical granuloma. 21 Balance between M1 and M2 is crucial in osteoblast differentiation derived from mesenchymal stem cell, which also affects the decay of bone homeostasis and regeneration during chronic inflammation. 19,22 Therefore, macrophages, especially polarized macrophages and macrophage-derived cytokines, are potential targets for the immunomodulation and improvement of AP. 16,21 However, the conditions underlying macrophage polarization in AP tissues remain controversial. 4,23,24 Pro-inflammatory cytokine IL-6 is instrumental in chronic inflammatory diseases and autoimmune diseases, as well as bone metabolism, and predominantly exerts its effect through the IL-6-signal transducer and activator of transcription 3 (STAT3) pathway. IL-6 transcription is induced by various stimuli, such as TLR ligands, IL-1, and TNF-α. 25 P.
gingivalis infection can disrupt oral biofilm balance and induce host responses that lead to overexpression of IL-6 and subsequent destruction of periodontal tissues. 22,26 Studies in oral diseases have speculated that IL-6 is an important marker for periodontitis. 27,28 Blocking the IL-6/STAT3 signaling pathway using the novel nonpeptidic inhibitor Stattic demonstrated promising potential in counteracting osteoclast differentiation and bone resorption in osteoarthritis. 29 However, the involvement of the IL-6/STAT3 pathway in the initiation and progression of AP, another oral lytic bone disease, has yet to be explored. Activation of the IL-6/STAT3 pathway is also reported to drive macrophages toward M2 differentiation in the tumor microenvironment. 25 Nevertheless, as a chemokine highly expressed by M1 macrophages, the effects of IL-6 and the related IL-6/STAT3 pathway on macrophage polarization in the AP context remains to be elucidated.

Ethics statement
Bacteria were collected when the culture reached an optical density at 600 nm of approximately 1.0 (~10 9 CFU mL -1 ) and were used for infection of unprimed

RT-qPCR
The generated gDNAs and cDNAs were subjected to RT-qPCR analysis performed on the Applied Biosystems Construction of a P. gingivalis-derived murine AP model P. gingivalis-derived AP was induced in mice according to methods described in Furusho's work. 32 The sample size was determined using G*power (version 3.1.9.7) (https://stats.idre.ucla.edu/other/ gpower/, UCLA, USA). 33,34 The sample size was estimated at 12 mice per group in order to achieve a P value less than 0.05 with an actual power of over 80%. A total of 48 8-week-old SPF male C57BL/6J mice (Dashuo Experimental Animals Co. Ltd, Chengdu, China) were included in the study. The mice were kept in a SPF environment with a 12 h light/dark cycle and given free access to food and water.
Mice were randomly assigned to different treatments (n=12 per group): no-treatment control (Con group); apical periodontitis (AP group); Stattic treatment (Stattic group); or 0.5% carboxymethylcellulose (Selleck) vehicle treatment (CMC group). AP was induced in all but the Con group. Briefly, after general anesthesia was induced by intraperitoneal injection of 0.4% sodium pentobarbital (J&K Scientific, Beijing, China), the operating area were disinfected with 75% ethanol containing moist sterile cotton swabs, special care was taken to prevent aspiration. Disinfectant was air-dried, then the pulp chamber of the mandibular first molar was exposed using a sterile #1/2 round drill to a depth equal to the diameter of the drill to prevent furcal perforation, with the process performed with a head mount magnifier loupe (Zeiss Inc, Thornwood, NY, USA). Next, a small sterile cotton swab, fully soaked with concentrated P. gingivalis W83 PBS suspension at a concentration of 1×10 9 CFU mL -1 , was applied in direct contact with the dental pulp for 5 min.

Statistical analysis
Experimental data were representative of three independent experiments with at least three samples.
For the TSA staining slices, 3 independent fields (at the magnification of × 600) from each sample were randomly selected and analyzed for MFI with ImageJ (https://imagej.net/software/fiji/downloads). The IL-6/STAT3 pathway was highly activated in human AP tissues infected by P. gingivalis highly expressed in AP tissues (p<0.001) ( Figure   2A and B) when compared with the healthy tissues.
RT-qPCR analysis revealed that the gene encoding IL-6 was also significantly induced in AP tissues   Figure 3A). This upregulation of IL-6 was confirmed by ELISA with a 4.6-fold average increase of IL-6 in the cell culture supernatant after 12 h of P. gingivalis infection (p<0.0001) ( Figure 3B).
Similar to the main signaling factor downstream of IL-6, the mRNA expression level of STAT3 remained stable upon stimulation with P. gingivalis (p>0.05) ( Figure   3C). However, the expression of phosphorylated STAT3 increased significantly after 12 h of P. gingivalis infection (p<0.01) ( Figure 3D and E), but decreased sharply after co-culturing with P. gingivalis for 24 h (data not shown). As p-STAT3 is the main effector of the IL-6/STAT3 pathway, 12 h -the point at which p-STAT3 expression peaked -was selected as the co-incubation period of P. gingivalis and BMDMs. The different trends of STAT3 mRNA and protein expression indicated that the phosphorylation status of STAT3, rather than the gene expression level of STAT3, was involved in the P. gingivalis-induced macrophage response.
IL-6 is one of the markers of the M1 macrophage phenotype. 17 Therefore, polarization of BMDMs, with or without P. gingivalis engagement, was observed using FC. P. gingivalis stimulation for 12 h promoted 54.4±11.9% of BMDMs to differentiate towards the M1 phenotype (CD86 + , as shown in quarters Q2 and Q3 of Figure 3F and G) (p<0.05), whereas the M2 type of differentiation was not significantly induced (CD206 + , as evidenced in Q1 and Q2 of Figure 3F and H) (p>0.05). Other M1 differentiation markers, including IL-1β and TNF-α, were also significantly  Histological analysis ( Figure 5C) indicated that the pulpal access was successfully generated without damaging the floor of the pulp chamber and also showed that the soft tissue surrounding the apical third of the teeth of the AP and CMC groups were more abundant when compared with those of the Con and Stattic groups, taken together with the CBCT scanning results, suggesting that apical bone  including TLRs, and protease-activated receptors (PARs), leading to multifunctional and/or phenotypic changes of macrophages. 18 The autocrine origin of the IL-6 in our experiment might also be a potential motivator that drives macrophages toward the M1, rather than the M2, phenotype. The murine AP model in the current study showed that the M1 phenotype of macrophages was associated with more significant bone lesions. The

Conclusions
The presence of M1 macrophages and IL-6/STAT3 expression was increased in lesion tissues of patients with chronic AP. P. gingivalis stimulation robustly induced IL-6 expression and STAT3 activation in BMDMs, and activation of the IL-6/STAT3 pathway mediated P. gingivalis-derived M1 polarization of BMDMs. P. gingivalis mono-species infection could cause detectable AP, recruiting macrophages to the apical region and directing their M1 polarization.
The P. gingivalis-infected BMDMs may accelerate bone destruction via secretion of IL-6, TNF-α, and RANKL, which hinder osteogenesis and promote osteoclastogenesis. The IL-6/STAT3 pathway is involved in mediating M1 polarization in the apical region and production of pro-inflammatory cytokines from P. gingivalis-infected BMDMs.