Nel-like Molecule Type 1 Combined with Gold Nanoparticles Modulates Macrophage Polarization, Osteoclastogenesis, and Oral Microbiota in Periodontitis

The disruption of host–microbe homeostasis and uncontrolled inflammatory response have been considered as vital causes for developing periodontitis, subsequently leading to an imbalance between the bone and immune system and the collapse of bone homeostasis. Consequently, strategies to modulate the immune response and bone metabolization have become a promising approach to prevent and treat periodontitis. In this study, we investigated the cooperative effects of Nel-like molecule type 1 (Nell-1) and gold nanoparticles (AuNPs) on macrophage polarization, osteoclast differentiation, and the corresponding functions in an experimental model of periodontitis in rats. Nell-1-combined AuNPs in in vitro studies were found to reduce the production of inflammatory factors (TNF-α, p p = 0.0012), modulate the ratio of M2/M1 macrophages by inducing macrophage polarization into the M2 phenotype, and inhibit cell fusion, maturation, and activity of osteoclasts. Furthermore, the local application of Nell-1-combined AuNPs in in vivo studies resulted in alleviation of damages to the periodontal and bone tissues, modulation of macrophage polarization and the activity of osteoclasts, and alteration of the periodontal microbiota, in which the relative abundance of the probiotic Bifidobacterium increased (p < 0.05). These findings reveal that Nell-1-combined AuNPs could be a promising drug candidate for the prevention and treatment of periodontitis. However, Nell-1-combined AuNPs did not show organ toxicity or impair the integrity of intestinal epithelium but alter the gut microbiota, leading to the dysbiosis of gut microbiota. The adverse impact of changes in gut microbiota needs to be further investigated. Nonetheless, this study provides a novel perspective and direction for the biological safety assessment of biomaterials in oral clinical applications

Additional file 1 of Association of per- and polyfluoroalkyl substances (PFAS) with periodontitis: the mediating role of sex hormones

Supplementary Material

P₄ suppresses E₂-dependent inflammatory responses in the ectopic lesions.

<p>Ectopic lesions were harvested from E₂ or E₂ plus P₄-treated recipients (n = 6) by 16 days after induction. (A) Representative images (20X) showing IHC analysis using antibodies against pan-macrophage biomarker (F4/80), inflammatory M1 (CCR7), anti-inflammatory M2 (CD206) macrophages or Treg cell biomarker (FOXP3), respectively. (B) The relative level of mRNA expression corresponding to <i>Ccl2</i>, <i>Ccl5</i>, <i>Il1b</i>, <i>Il6</i>, <i>Tnfa</i>, and <i>Tgfb</i> was analyzed by qPCR after moralization to the internal control gene, <i>36B4</i>. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.</p

Immunocompetent mouse model of endometriosis.

<p>6–8 weeks-old CD1 female mice were primed with PMSG for 48 hours. Uterine tissues were then harvested and minced into tiny cell aggregates after myometrial removal. Female mice with the same genetic background were subjected to ovariectomy and served as recipients. 2 weeks after surgery, equal volumes of uterine cell aggregate suspension were transferred into the peritoneal cavities of recipients. Endometriosis was maintained by subcutaneous administration of 100 ng of E₂ once every 4 days until tissue collection. For the studies of the role of P₄ in endometriosis, 1 mg of P₄ (pre-P₄) was administrated along with E₂ beginning at 4 days before transplantation. For P4-resistance experiments (Post-P₄), 1 mg of P₄ was administration along with E2 beginning at 4 days after transplantation. The ectopic lesions were assessed under a dissecting microscope at different days after endometrial cell transplantation (n = 6 per treatment group).</p

P₄ alleviates E₂-dependent establishment and growth of ectopic lesions.

<p>Ectopic lesions were established in the peritoneal cavities of immunocompetent female mice and treated with E₂ or P₄ along with E₂ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165347#pone.0165347.g001" target="_blank">Fig 1</a> (N = 6). (A) Locations of ectopic lesions (Dashed circles). (B) Histology of ectopic lesions. Dashed lines indicate the contact sites of the ectopic lesions with adjacent peritoneum. Representative images of ectopic lesions harvested at four weeks after induction are shown (40X). (C) Time-course progression of ectopic lesions. The average volumes of the ectopic lesions at the indicated time points are shown. (D) The average numbers of ectopic lesions identified in each treatment group are shown. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as ^#: p < 0.05, *: p<0.01.</p

P₄ inhibits E₂-dependent cell proliferation and angiogenesis in ectopic lesions.

<p>Sections of the ectopic lesions collected from E₂ or E₂ plus P₄-treated recipients (D16, n = 6) were subjected to histological examination. (A) Representative images (20X) showing H&E and Trichrome staining, or IHC analysis using antibody against myofibroblast biomarker αSMA, uterine epithelial biomarker KRT11, or uterine stromal biomarker VIM, respectively. (B) Representative images (20X) showing IHC analysis using antibodies against cell proliferation biomarker KI67, smooth muscle biomarker αSMA, endothelial cells CD31, or an angiogenetic regulator CCN1, respectively. The numbers of KI67-positive cells, the perimeters of the supporting blood vessels, and the immunostaining intensities of CCN1 and CD31 were analyzed by ImageJ software. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.</p

Loss of ERα/PR-mediated signaling contributes to P₄-resistance in this mouse model of endometriosis.

<p>Endometriosis was induced and maintained with E₂ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165347#pone.0165347.g001" target="_blank">Fig 1</a>. The host females were then treated with P₄ beginning at 4 days before (Pre-) or 4 days after (Post-) endometrial cell transplantation until tissue collection (n = 6). Donor uterine tissue (D0) and ectopic lesions were subjected to IHC analysis (A) for ERα, PR and HAND2 protein expression (20X) or qPCR analysis (B) to assess expression level of mRNA corresponding to <i>Esr1</i>, <i>Pgr</i>, <i>Hand2</i>, and <i>Hoxa10</i>, respectively. (C) Lesion volumes were quantitated by 16 days after induction. The numerical values were analyzed by One-way ANOVA followed by Dunnett’s post hoc test and expressed as mean ± SEM. Statistical significance is defined as #: p < 0.05, *: p<0.01.</p

Surveillance of Cancer Stem Cell Plasticity Using an Isoform-Selective Fluorescent Probe for Aldehyde Dehydrogenase 1A1

Cancer stem cells (CSCs) are progenitor cells that contribute to treatment-resistant phenotypes during relapse. CSCs exist in specific tissue microenvironments that cell cultures and more complex models cannot mimic. Therefore, the development of new approaches that can detect CSCs and report on specific properties (e.g., stem cell plasticity) in their native environment have profound implications for studying CSC biology. Herein, we present AlDeSense, a turn-on fluorescent probe for aldehyde dehydrogenase 1A1 (ALDH1A1) and Ctrl-AlDeSense, a matching nonresponsive reagent. Although ALDH1A1 contributes to the detoxification of reactive aldehydes, it is also associated with stemness and is highly elevated in CSCs. AlDeSense exhibits a 20-fold fluorescent enhancement when treated with ALDH1A1. Moreover, we established that AlDeSense is selective against a panel of common ALDH isoforms and exhibits exquisite chemostability against a collection of biologically relevant species. Through the application of surface marker antibody staining, tumorsphere assays, and assessment of tumorigenicity, we demonstrate that cells exhibiting high AlDeSense signal intensity have properties of CSCs. Using these probes in tandem, we have identified CSCs at the cellular level via flow cytometry and confocal imaging, as well as monitored their states in animal models