Epithelial-derived interleukin-23 promotes oral mucosal immunopathology.

At mucosal surfaces, epithelial cells provide a structural barrier and an immune defense system. However, dysregulated epithelial responses can contribute to disease states. Here, we demonstrated that epithelial cell-intrinsic production of interleukin-23 (IL-23) triggers an inflammatory loop in the prevalent oral disease periodontitis. Epithelial IL-23 expression localized to areas proximal to the disease-associated microbiome and was evident in experimental models and patients with common and genetic forms of disease. Mechanistically, flagellated microbial species of the periodontitis microbiome triggered epithelial IL-23 induction in a TLR5 receptor-dependent manner. Therefore, unlike other Th17-driven diseases, non-hematopoietic-cell-derived IL-23 served as an initiator of pathogenic inflammation in periodontitis. Beyond periodontitis, analysis of publicly available datasets revealed the expression of epithelial IL-23 in settings of infection, malignancy, and autoimmunity, suggesting a broader role for epithelial-intrinsic IL-23 in human disease. Collectively, this work highlights an important role for the barrier epithelium in the induction of IL-23-mediated inflammation.

Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A.

Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity. Treatment with the siRNA-carrying LNPs reduced circulating plasminogen and suppressed fibrinolysis in wild-type and HA mice and dogs. In HA mice, hemostatic efficacy depended on the injury model; plasminogen knockdown improved hemostasis after a saphenous vein injury but not tail vein transection injury, suggesting that saphenous vein injury is a murine bleeding model sensitive to the contribution of fibrinolysis. In dogs with HA, LNPs carrying siRNA targeting plasminogen were as effective at stabilizing clots as tranexamic acid, a clinical antifibrinolytic, and in a pilot study of two dogs with HA, the incidence of spontaneous or excess bleeding was reduced during 4 months of prolonged knockdown. Collectively, these data demonstrate that long-acting antifibrinolytic therapy can be achieved and that it provides hemostatic benefit in animal models of HA.

Periodontal disease in patients with WHIM syndrome.

AIM: WHIM (warts, hypogammaglobulinaemia, infections and myelokathexis) syndrome is a rare combined primary immunodeficiency disease caused by gain-of-function (GOF) mutations in the chemokine receptor CXCR4 and includes severe neutropenia as a common feature. Neutropenia is a known risk factor for periodontitis; however, a detailed periodontal evaluation of a WHIM syndrome cohort is lacking. This study aimed to establish the evidence base for the periodontal status of patients with WHIM syndrome. MATERIALS AND METHODS: Twenty-two adult WHIM syndrome patients and 22 age- and gender-matched healthy volunteers (HVs) were evaluated through a comprehensive medical and periodontal examination. A mouse model of WHIM syndrome was assessed for susceptibility to naturally progressing or inducible periodontitis. RESULTS: Fourteen patients with WHIM syndrome (63.6%) and one HV (4.5%) were diagnosed with Stage III/IV periodontitis. No WHIM patient presented with the early onset, dramatic clinical phenotypes typically associated with genetic forms of neutropenia. Age, but not the specific CXCR4 mutation or absolute neutrophil count, was associated with periodontitis severity in the WHIM cohort. Mice with a Cxcr4 GOF mutation did not exhibit increased alveolar bone loss in spontaneous or ligature-induced periodontitis. CONCLUSIONS: Overall, WHIM syndrome patients presented with an increased severity of periodontitis despite past and ongoing neutrophil mobilization treatments. GOF mutations in CXCR4 may be a risk factor for periodontitis in humans.

Neutrophil extracellular traps and extracellular histones potentiate IL-17 inflammation in periodontitis.

Neutrophil infiltration is a hallmark of periodontitis, a prevalent oral inflammatory condition in which Th17-driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model, we demonstrate that neutrophils infiltrate the gingival oral mucosa at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanisms by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL-17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document significantly increased levels of NET complexes and extracellular histones bearing classic NET-associated posttranslational modifications, in blood and local lesions of severe periodontitis patients, in the absence of confounding disease. Our findings suggest a feed-forward loop in which NETs trigger IL-17 immunity to promote immunopathology in a prevalent human inflammatory disease.

C5a-licensed phagocytes drive sterilizing immunity during systemic fungal infection.

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.

Neutrophils are gatekeepers of mucosal immunity.

Mucosal tissues are constantly exposed to the outside environment. They receive signals from the commensal microbiome and tissue-specific triggers including alimentary and airborne elements and are tasked to maintain balance in the absence of inflammation and infection. Here, we present neutrophils as sentinel cells in mucosal immunity. We discuss the roles of neutrophils in mucosal homeostasis and overview clinical susceptibilities in patients with neutrophil defects. Finally, we present concepts related to specification of neutrophil responses within specific mucosal tissue microenvironments.

Osteoimmunology in periodontitis; a paradigm for Th17/IL-17 inflammatory bone loss.

Periodontitis is a prevalent human disease of inflammation-induced bone destruction. Through studies in patient lesions of rare and common forms of periodontitis and animal model experimentation, Th17/IL-17 related immune pathways have emerged as mediators of disease pathology. In this focused review, we examine mechanisms of induction, amplification and pathogenicity of Th17 cells in periodontitis.

Regional specification of oral mucosal immunity.

In this issue of Science Immunology, Barreto de Albuquerque et al. track immune responsiveness to the foodborne pathogen Listeria monocytogenes during oral infection. Their findings extend the notion of compartmentalized immunity within the gastrointestinal tract to the oral cavity and provide previously unkown insights into regional specialization of oral immunity.

Alterations of human skin microbiome and expansion of antimicrobial resistance after systemic antibiotics.

Although systemic antibiotics are critical in controlling infections and reducing morbidity and mortality, overuse of antibiotics is presumed to contribute to negative repercussions such as selection of antimicrobial-resistant organisms and collateral damage to commensal microbes. In a prospective, randomized study of four clinically relevant antibiotic regimens [doxycycline (20 mg or 100 mg), cephalexin, or trimethoprim/sulfamethoxazole], we investigated microbial alterations on skin after administration of systemic antibiotics to healthy human volunteers. Samples from different skin and oral sites, as well as stool, were collected before, during, and up to 1 year after antibiotic use, and shotgun metagenomic sequencing was performed. Taxonomic analysis showed that subjects receiving doxycycline 100 mg and trimethoprim/sulfamethoxazole (TMP/SMX) exhibited greater changes to their skin microbial communities, as compared to those receiving other regimens or untreated controls. Oral and stool microbiota also demonstrated fluctuations after antibiotics. Bacterial culturing in combination with whole-genome sequencing revealed specific emergence, expansion, and persistence of antibiotic-resistant staphylococci harboring tetK or tetL and dfrC or dfrG genes in all subjects who received doxycycline 100 mg or TMP/SMX, respectively. Last, analysis of metagenomic data revealed an increase of genes involved in gene mobilization, indicating stress responses of microbes to antibiotics. Collectively, these findings demonstrate direct, long-lasting effects of antibiotics on skin microbial communities, highlighting the skin microbiome as a site for the development and persistence of antibiotic resistance and the risks of overprescribing.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the αMß2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG, encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

Response to Comments on "Aberrant type 1 immunity drives susceptibility to mucosal fungal infections".

Puel and Casanova and Kisand et al. challenge our conclusions that interferonopathy and not IL-17/IL-22 autoantibodies promote candidiasis in autoimmune polyendocrinopathy­candidiasis­ectodermal dystrophy. We acknowledge that conclusive evidence for causation is difficult to obtain in complex human diseases. However, our studies clearly document interferonopathy driving mucosal candidiasis with intact IL-17/IL-22 responses in Aire-deficient mice, with strong corroborative evidence in patients.

Infections in the monogenic autoimmune syndrome APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by mutations in the Autoimmune Regulator (AIRE) gene, which impair the thymic negative selection of self-reactive T-cells and underlie the development of autoimmunity that targets multiple endocrine and non-endocrine tissues. Beyond autoimmunity, APECED features heightened susceptibility to certain specific infections, which is mediated by anti-cytokine autoantibodies and/or T-cell driven autoimmune tissue injury. These include the 'signature' APECED infection chronic mucocutaneous candidiasis (CMC), but also life-threatening coronavirus disease 2019 (COVID-19) pneumonia, bronchiectasis-associated bacterial pneumonia, and sepsis by encapsulated bacteria. Here we discuss the expanding understanding of the immunological mechanisms that contribute to infection susceptibility in this prototypic syndrome of impaired central tolerance, which provide the foundation for devising improved diagnostic and therapeutic strategies for affected patients.

Aberrant type 1 immunity drives susceptibility to mucosal fungal infections.

Human monogenic disorders have revealed the critical contribution of type 17 responses in mucosal fungal surveillance. We unexpectedly found that in certain settings, enhanced type 1 immunity rather than defective type 17 responses can promote mucosal fungal infection susceptibility. Notably, in mice and humans with AIRE deficiency, an autoimmune disease characterized by selective susceptibility to mucosal but not systemic fungal infection, mucosal type 17 responses are intact while type 1 responses are exacerbated. These responses promote aberrant interferon-γ (IFN-γ)- and signal transducer and activator of transcription 1 (STAT1)-dependent epithelial barrier defects as well as mucosal fungal infection susceptibility. Concordantly, genetic and pharmacologic inhibition of IFN-γ or Janus kinase (JAK)-STAT signaling ameliorates mucosal fungal disease. Thus, we identify aberrant T cell-dependent, type 1 mucosal inflammation as a critical tissue-specific pathogenic mechanism that promotes mucosal fungal infection susceptibility in mice and humans.

A cross-species interaction with a symbiotic commensal enables cell-density-dependent growth and in vivo virulence of an oral pathogen.

Recent studies describe in detail the shifts in composition of human-associated polymicrobial communities from health to disease. However, the specific processes that drive the colonization and overgrowth of pathogens within these communities remain incompletely understood. We used in vitro culture systems and a disease-relevant mouse model to show that population size, which determines the availability of an endogenous diffusible small molecule, limits the growth, colonization, and in vivo virulence of the human oral pathogen Porphyromonas gingivalis. This bacterial pathogen overcomes the requirement for an endogenous cue by utilizing a cell-density dependent, growth-promoting, soluble molecule provided by the symbiotic early colonizer Veillonella parvula, but not produced by other commensals tested. Our work shows that exchange of cell-density-dependent diffusible cues between specific early and late colonizing species in a polymicrobial community drives microbial successions, pathogen colonization and disease development, representing a target process for manipulation of the microbiome towards the healthy state.

Case Report: Dental Findings Can Aid in Early Diagnosis of APECED Syndrome.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also known as autoimmune polyglandular syndrome type 1 (APS-1), is a rare genetic disorder caused most often by biallelic mutations in the AIRE gene. Classic clinical findings of the disease are chronic mucocutaneous candidiasis and autoimmunity that primarily targets endocrine tissues, such as hypoparathyroidism and adrenal insufficiency. Recently, however, it has been appreciated that enamel hypoplasia, together with intestinal malabsorption and a characteristic APECED rash, is a prominent early disease manifestation of APECED which can aid in the diagnosis of disease before other potentially life-threatening disease manifestations occur. To demonstrate this point, we present data from a cohort of APECED patients, approximately 70% of who present with enamel dysplasia at an early age. Importantly, early life presentation with enamel dysplasia was predictive of likelihood of development of a subsequent APECED diagnosis. Furthermore, we present a case of a patient with APECED and severe enamel defects and discuss the utility of medical-dental professional co-operation in the diagnosis and management of this complex disorder.

T cell exosome-derived miR-142-3p impairs glandular cell function in Sjögren's syndrome.

Sjögren's syndrome (SS) is a systemic autoimmune disease that mainly affects exocrine salivary and lacrimal glands. Local inflammation in the glands is thought to trigger glandular dysfunction and symptoms of dryness. However, the mechanisms underlying these processes are incompletely understood. Our work suggests T cell exosome-derived miR-142-3p as a pathogenic driver of immunopathology in SS. We first document miR-142-3p expression in the salivary glands of patients with SS, both in epithelial gland cells and within T cells of the inflammatory infiltrate, but not in healthy volunteers. Next, we show that activated T cells secreted exosomes containing miR-142-3p, which transferred into glandular cells. Finally, we uncover a functional role of miR-142-3p-containing exosomes in glandular cell dysfunction. We find that miR-142-3p targets key elements of intracellular Ca2+ signaling and cAMP production - sarco(endo)plasmic reticulum Ca2+ ATPase 2b (SERCA2B), ryanodine receptor 2 (RyR2), and adenylate cyclase 9 (AC9) - leading to restricted cAMP production, altered calcium signaling, and decreased protein production from salivary gland cells. Our work provides evidence for a functional role of the miR-142-3p in SS pathogenesis and promotes the concept that T cell activation may directly impair epithelial cell function through secretion of miRNA-containing exosomes.

Frontline Science: Activation of metabolic nuclear receptors restores periodontal tissue homeostasis in mice with leukocyte adhesion deficiency-1.

ß2 Integrins mediate neutrophil-endothelial adhesion and recruitment of neutrophils to sites of inflammation. The diminished expression of ß2 integrins in patients with mutations in the ITGB2 (CD18) gene (leukocyte adhesion deficiency-Type 1; LAD1) results in few or no neutrophils in peripheral tissues. In the periodontium, neutrophil paucity is associated with up-regulation of IL-23 and IL-17, which drive inflammatory bone loss. Using a relevant mouse model, we investigated whether diminished efferocytosis (owing to neutrophil scarcity) is associated with LAD1 periodontitis pathogenesis and aimed to develop approaches to restore the missing efferocytosis signals. We first showed that CD18-/- mice phenocopied human LAD1 in terms of IL-23/IL-17-driven inflammatory bone loss. Ab-mediated blockade of c-Mer tyrosine kinase (Mer), a major efferocytic receptor, mimicked LAD1-associated up-regulation of gingival IL-23 and IL-17 mRNA expression in wild-type (WT) mice. Consistently, soluble Mer-Fc reversed the inhibitory effect of efferocytosis on IL-23 expression in LPS-activated Mϕs. Adoptive transfer of WT neutrophils to CD18-/- mice down-regulated IL-23 and IL-17 expression to normal levels, but not when CD18-/- mice were treated with blocking anti-Mer Ab. Synthetic agonist-induced activation of liver X receptors (LXR) and peroxisome proliferator-activated receptors (PPAR), which link efferocytosis to generation of homeostatic signals, inhibited the expression of IL-23 and IL-17 and favorably affected the bone levels of CD18-/- mice. Therefore, our data link diminished efferocytosis-associated signaling due to impaired neutrophil recruitment to dysregulation of the IL-23-IL-17 axis and, moreover, suggest LXR and PPAR as potential therapeutic targets for treating LAD1 periodontitis.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Regulation of host-microbe interactions at oral mucosal barriers by type 17 immunity.

The oral mucosa is a primary barrier site and a portal for entry of microbes, food, and airborne particles into the gastrointestinal tract. Nonetheless, mucosal immunity at this barrier remains understudied compared with other anatomical barrier sites. Here, we review basic aspects of oral mucosal histology, the oral microbiome, and common and clinically significant diseases that present at oral mucosal barriers. We particularly focus on the role of interleukin-17 (IL-17)/T helper 17 (TH17) responses in protective immunity and inflammation in the oral mucosa. IL-17/TH17 responses are highly relevant to maintaining barrier integrity and preventing pathogenic infections by the oral commensal fungus Candida albicans On the other hand, aberrant IL-17/TH17 responses are implicated in driving the pathogenesis of periodontitis and consequent bone and tooth loss. We discuss distinct IL-17-secreting T cell subsets, emphasizing their regulation and function in oropharyngeal candidiasis and periodontitis.

Plasmin-mediated fibrinolysis enables macrophage migration in a murine model of inflammation.

Efficient migration of macrophages to sites of inflammation requires cell surface-bound plasmin(ogen). Here, we investigated the mechanisms underlying the deficits of plasmin(ogen)-mediated macrophage migration in 2 models: murine thioglycollate-induced peritonitis and in vitro macrophage migration. As previously reported, macrophage migration into the peritoneal cavity of mice in response to thioglycollate was significantly impaired in the absence of plasminogen. Fibrin(ogen) deposition was noted in the peritoneal cavity in response to thioglycollate, with a significant increase in fibrin(ogen) in the plasminogen-deficient mice. Interestingly, macrophage migration was restored in plasminogen-deficient mice by simultaneous imposition of fibrinogen deficiency. Consistent with this in vivo finding, chemotactic migration of cultured macrophages through a fibrin matrix did not occur in the absence of plasminogen. The macrophage requirement for plasmin-mediated fibrinolysis, both in vivo and in vitro, was negated by deletion of the major myeloid integrin αMß2-binding motif on the γ chain of fibrin(ogen). The study identifies a critical role of fibrinolysis in macrophage migration, presumably through the alleviation of migratory constraints imposed by the interaction of leukocytes with fibrin(ogen) through the integrin αMß2 receptor.