Mutual interplay between IL-17-producing γδT cells and microbiota orchestrates oral mucosal homeostasis.

γδT cells are a major component of epithelial tissues and play a role in tissue homeostasis and host defense. γδT cells also reside in the gingiva, an oral tissue covered with specialized epithelium that continuously monitors the challenging dental biofilm. Whereas most research on intraepithelial γδT cells focuses on the skin and intestine epithelia, our knowledge on these cells in the gingiva is still incomplete. In this study, we demonstrate that even though the gingiva develops after birth, the majority of gingival γδT cells are fetal thymus-derived Vγ6+ cells, and to a lesser extent Vγ1+ and Vγ4+ cells. Furthermore, we show that γδT cells are motile and locate preferentially in the epithelium adjacent to the biofilm. Vγ6+ cells represent the major source of IL-17-producing cells in the gingiva. Chimeric mice and parabiosis experiments indicated that the main fraction of gingival γδT cells is radioresistant and tissue-resident, persisting locally independent of circulating γδT cells. Notably, gingival γδT cell homeostasis is regulated by the microbiota as the ratio of Vγ6+ and Vγ4+ cells was reversed in germ-free mice, and their activation state was decreased. As a consequence, conditional ablation of γδT cells results in elevated gingival inflammation and subsequent alterations of oral microbial diversity. Taken together, these findings suggest that oral mucosal homeostasis is shaped by reciprocal interplays between γδT cells and local microbiota.

Microbiota-dependent and -independent postnatal development of salivary immunity.

While saliva regulates the interplay between the microbiota and the oral immune system, the mechanisms establishing postnatal salivary immunity are ill-defined. Here, we show that high levels of neutrophils and neonatal Fc receptor (FcRn)-transferred maternal IgG are temporarily present in the neonatal murine salivary glands in a microbiota-independent manner. During weaning, neutrophils, FcRn, and IgG decrease in the salivary glands, while the polymeric immunoglobulin receptor (pIgR) is upregulated in a growth arrest-specific 6 (GAS6)-dependent manner independent of the microbiota. Production of salivary IgA begins following weaning and relies on CD4-help, IL-17, and the microbiota. The weaning phase is characterized by a transient accumulation of dendritic cells capable of migrating from the oral mucosa to the salivary glands upon exposure to microbial challenges and activating T cells. This study reveals the postnatal mechanisms developed in the salivary glands to induce immunity and proposes the salivary glands as an immune inductive site.

Development and characterization of high affinity leptins and leptin antagonists.

Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

Maturation of the neonatal oral mucosa involves unique epithelium-microbiota interactions.

Postnatal host-microbiota interplay governs mucosal homeostasis and is considered to have life-long health consequences. The intestine monolayer epithelium is critically involved in such early-life processes; nevertheless, the role of the oral multilayer epithelium remains ill defined. We demonstrate that unlike the intestine, the neonate oral cavity is immensely colonized by the microbiota that decline to adult levels during weaning. Neutrophils are present in the oral epithelium prenatally, and exposure to the microbiota postnatally further recruits them to the preamble neonatal epithelium by γδT17 cells. These neutrophils virtually disappear during weaning as the epithelium seals. The neonate and adult epithelium display distinct turnover kinetics and transcriptomic signatures, with neonate epithelium reminiscent of the signature found in germ-free mice. Microbial reduction during weaning is mediated by the upregulation of saliva production and induction of salivary antimicrobial components by the microbiota. Collectively, unique postnatal interactions between the multilayer epithelium and microbiota shape oral homeostasis.

Niche rather than origin dysregulates mucosal Langerhans cells development in aged mice.

Unlike epidermal Langerhans cells (LCs) that originate from embryonic precursors and are self-renewed locally, mucosal LCs arise and are replaced by circulating bone marrow (BM) precursors throughout life. While the unique lifecycle of epidermal LCs is associated with an age-dependent decrease in their numbers, whether and how aging has an impact on mucosal LCs remains unclear. Focusing on gingival LCs we found that mucosal LCs are reduced with age but exhibit altered morphology with that observed in aged epidermal LCs. The reduction of gingival but not epidermal LCs in aged mice was microbiota-dependent; nevertheless, the impact of the microbiota on gingival LCs was indirect. We next compared the ability of young and aged BM precursors to differentiate to mucosal LCs. Mixed BM chimeras, as well as differentiation cultures, demonstrated that aged BM has intact if not superior capacity to differentiate into LCs than young BM. This was in line with the higher percentages of mucosal LC precursors, pre-DCs, and monocytes, detected in aged BM. These findings suggest that while aging is associated with reduced LC numbers, the niche rather than the origin controls this process in mucosal barriers.

Leukocyte-specific siRNA delivery revealing IRF8 as a potential anti-inflammatory target.

Interferon regulatory factor 8 (IRF8) protein plays a critical role in the differentiation, polarization, and activation of mononuclear phagocytic cells. In light of previous studies, we explored the therapeutic potential of IRF8 inhibition as immunomodulatory therapy for inflammatory bowel disease (IBD). To this end, we utilized siRNA-loaded lipid-based nanoparticles (siLNPs) and demonstrated a ∼90% reduction of IRF8 mRNA levels in vitro (PV < 0.0001), alongside a notable reduction in IRF8 protein. Moreover, silencing IRF8 ex vivo in splenocytes lead to a profound downregulation of IRF8 protein, followed by an immunomodulatory effect, as represented by a decrease in the secretion of TNFα, IL6 and IL12/IL23 (IL12p40) proinflammatory cytokines (PV = 0.0045, 0.0330, <0.0001, respectively). In order to silence IRF8 in vivo, selectively in inflammatory leukocytes, we used siLNPs that were coated with anti-Ly6C antibodies via our recently published ASSET targeting approach. Through this strategy, we have demonstrated a selective binding of the targeted-LNPs (T-LNPs) to Ly6C + inflammatory leukocytes. Finally, an immunomodulatory effect was demonstrated in vivo in an IBD mouse model with a profound decrease of TNFα, IL6, IL12/IL23, and IL1ß pro-inflammatory cytokines (n = 5, PV < 0.0001, <0.0001, <0.0001, 0.02, respectively) and an improvement of colon-morphology as assessed by colon-length measurements and colonoscopy (PV < 0.0001). Overall, using antibody-targeted siLNPs, we showed a notable reduction of IRF8 mRNA and protein and demonstrated a targeted immunomodulation therapeutic effect ex vivo and in vivo, in the DSS colitis model. We claim that a selective silencing of IRF8 in inflammatory leukocytes (such as Ly6C+) may serve as a therapeutic approach for treating inflammatory disorders.

Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation.

Intestinal colonization by bacteria of oral origin has been correlated with several negative health outcomes, including inflammatory bowel disease. However, a causal role of oral bacteria ectopically colonizing the intestine remains unclear. Using gnotobiotic techniques, we show that strains of Klebsiella spp. isolated from the salivary microbiota are strong inducers of T helper 1 (TH1) cells when they colonize in the gut. These Klebsiella strains are resistant to multiple antibiotics, tend to colonize when the intestinal microbiota is dysbiotic, and elicit a severe gut inflammation in the context of a genetically susceptible host. Our findings suggest that the oral cavity may serve as a reservoir for potential intestinal pathobionts that can exacerbate intestinal disease.

Pegylated leptin antagonist is a potent orexigenic agent: preparation and mechanism of activity.

Leptin, a pleiotropic adipokine, is a central regulator of appetite and weight and a key immunomodulatory protein. Although inborn leptin deficiency causes weight gain, it is unclear whether induced leptin deficiency in adult wild-type animals would be orexigenic. Previous work with a potent competitive leptin antagonist did not induce a true metabolic state of leptin deficiency in mice because of a short circulating half-life. In this study, we increased the half-life of the leptin antagonist by pegylation, which resulted in significantly increased bioavailability and retaining of antagonistic activity. Mice administered the pegylated antagonist showed a rapid and dramatic increase in food intake with weight gain. Resulting fat was confined to the mesenteric region with no accumulation in the liver. Serum cholesterol, triglyceride, and hepatic aminotransferases remained unaffected. Weight changes were reversible on cessation of leptin antagonist treatment. The mechanism of severe central leptin deficiency was found to be primarily caused by blockade of transport of circulating leptin across the blood-brain barrier with antagonisms at the arcuate nucleus playing a more minor role. Altogether we introduce a novel compound that induces central and peripheral leptin deficiency. This compound should be useful in exploring the involvement of leptin in metabolic and immune processes and could serve as a therapeutic for the treatment of cachexia.