Adolescence and the Microbiome: Implications for Healthy Growth and Maturation.

The gut microbiota was initially thought to develop into a stable, adult-like profile during early postnatal life. The formation of the gut microbiota during early life has been shown to contribute to healthy growth and has lifelong implications for host health. Adolescence, the developmental period between childhood and adulthood, is a critical window for healthy growth and maturation. The composition of the gut microbiota in adolescents is distinct from that of children and adults, which supports the premise that the gut microbiota continues to develop during adolescence toward an adult-like profile. Research has begun to shift its focus from understanding the gut microbiome at the extremes of the life span to evaluating the importance of the gut microbiome during adolescence and its role in healthy development. This article provides an overview of adolescent development, host-microbiota interactions, and experimental models used to discern effects of gut microbiota on health and disease. Herein, the role of the gut microbiota is reviewed as it relates to adolescent: i) brain development, cognition, and behavior; ii) metabolism and adiposity; and iii) skeletal growth and bone mass accrual. Future directions are addressed, including omics investigations defining mechanisms through which the gut microbiota influences adolescent development. Furthermore, we discuss advancing noninvasive interventions targeting the adolescent gut microbiota that could be employed to support healthy growth and maturation.

Prolonged Antibiotic Exposure during Adolescence Dysregulates Liver Metabolism and Promotes Adiposity in Mice.

Antibiotic administration during early life has been shown to have lasting effects on the gut microbiota, which have been linked to sustained alterations in liver metabolism and adiposity. Recent investigations have discerned that the gut microbiota continues to develop toward an adult-like profile during adolescence. However, the impact of antibiotic exposure during adolescence on metabolism and adiposity is unclear. Herein, a retrospective analysis of Medicaid claims data was performed, which indicated that tetracycline class antibiotics are commonly prescribed for the systemic treatment of adolescent acne. The purpose of this was to discern the impact of a prolonged tetracycline antibiotic exposure during adolescence on the gut microbiota, liver metabolism, and adiposity. Male C57BL/6T specific pathogen-free mice were administered a tetracycline antibiotic during the pubertal/postpubertal adolescent growth phase. Groups were euthanized at different time points to assess immediate and sustained antibiotic treatment effects. Antibiotic exposure during adolescence caused lasting genera-level shifts in the intestinal bacteriome and persistent dysregulation of metabolic pathways in the liver. Dysregulated hepatic metabolism was linked to sustained disruption of the intestinal farnesoid X receptor-fibroblast growth factor 15 axis, a gut-liver endocrine axis that supports metabolic homeostasis. Antibiotic exposure during adolescence increased subcutaneous, visceral, and marrow adiposity, which intriguingly manifested following antibiotic therapy. This preclinical work highlights that prolonged antibiotic courses for the clinical treatment of adolescent acne may have unintended deleterious effects on liver metabolism and adiposity.

Commensal gut bacterium critically regulates alveolar bone homeostasis.

The alveolar bone is a unique osseous tissue due to the presence of the teeth and the proximity of commensal oral microbes. Commensal microbe effects on alveolar bone homeostasis have been attributed to the oral microbiota, yet the impact of commensal gut microbes is unknown. Study purpose was to elucidate whether commensal gut microbes regulate osteoimmune mechanisms and skeletal homeostasis in alveolar bone. Male C57BL/6T germfree (GF) littermate mice were maintained as GF or monoassociated with segmented filamentous bacteria (SFB), a commensal gut bacterium. SFB has been shown to elicit broad immune response effects, including the induction of TH17/IL17A immunity, which impacts the development and homeostasis of host tissues. SFB colonized the gut, but not oral cavity, and increased IL17A levels in the ileum and serum. SFB had catabolic effects on alveolar bone and non-oral skeletal sites, which was attributed to enhanced osteoclastogenesis. The alveolar bone marrow of SFB vs. GF mice had increased dendritic cells, activated helper T-cells, TH1 cells, TH17 cells, and upregulated Tnf. Primary osteoblast cultures from SFB and GF mice were stimulated with vehicle-control, IL17A, or TNF to elucidate osteoblast-derived signaling factors contributing to the pro-osteoclastic phenotype in SFB mice. Treatment of RAW264.7 osteoclastic cells with supernatants from vehicle-stimulated SFB vs. GF osteoblasts recapitulated the osteoclast phenotype found in vivo. Supernatants from TNF-stimulated osteoblasts normalized RAW264.7 osteoclast endpoints across SFB and GF cultures, which was dependent on the induction of CXCL1 and CCL2. This report reveals that commensal gut microbes have the capacity to regulate osteoimmune processes in alveolar bone. Outcomes from this investigation challenge the current paradigm that alveolar bone health and homeostasis is strictly regulated by oral microbes.

Antimicrobial-induced oral dysbiosis exacerbates naturally occurring alveolar bone loss.

Periodontitis-mediated alveolar bone loss is caused by dysbiotic shifts in the commensal oral microbiota that upregulate proinflammatory osteoimmune responses. The study purpose was to determine whether antimicrobial-induced disruption of the commensal microbiota has deleterious effects on alveolar bone. We administered an antibiotic cocktail, minocycline, or vehicle-control to sex-matched C57BL/6T mice from age 6- to 12 weeks. Antibiotic cocktail and minocycline had catabolic effects on alveolar bone in specific-pathogen-free (SPF) mice. We then administered minocycline or vehicle-control to male mice reared under SPF and germ-free conditions, and we subjected minocycline-treated SPF mice to chlorhexidine oral antiseptic rinses. Alveolar bone loss was greater in vehicle-treated SPF versus germ-free mice, demonstrating that the commensal microbiota drives naturally occurring alveolar bone loss. Minocycline- versus vehicle-treated germ-free mice had similar alveolar bone loss outcomes, implying that antimicrobial-driven alveolar bone loss is microbiota dependent. Minocycline induced phylum-level shifts in the oral bacteriome and exacerbated naturally occurring alveolar bone loss in SPF mice. Chlorhexidine further disrupted the oral bacteriome and worsened alveolar bone loss in minocycline-treated SPF mice, validating that antimicrobial-induced oral dysbiosis has deleterious effects on alveolar bone. Minocycline enhanced osteoclast size and interface with alveolar bone in SPF mice. Neutrophils and plasmacytoid dendritic cells were upregulated in cervical lymph nodes of minocycline-treated SPF mice. Paralleling the upregulated proinflammatory innate immune cells, minocycline therapy increased TH 1 and TH 17 cells that have known pro-osteoclastic actions in the alveolar bone. This report reveals that antimicrobial perturbation of the commensal microbiota induces a proinflammatory oral dysbiotic state that exacerbates naturally occurring alveolar bone loss.

Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice.

Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ-free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin-induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif-deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild-type mice. Isolated Myd88/Trif-deficient hepatocytes were more susceptible to toxin-induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis.

Commensal Microbiota Effects on Craniofacial Skeletal Growth and Morphology.

Microbes colonize anatomical sites in health to form commensal microbial communities (e.g., commensal gut microbiota, commensal skin microbiota, commensal oral microbiota). Commensal microbiota has indirect effects on host growth and maturation through interactions with the host immune system. The commensal microbiota was recently introduced as a novel regulator of skeletal growth and morphology at noncraniofacial sites. Further, we and others have shown that commensal gut microbes, such as segmented filamentous bacteria (SFB), contribute to noncraniofacial skeletal growth and maturation. However, commensal microbiota effects on craniofacial skeletal growth and morphology are unclear. To determine the commensal microbiota's role in craniofacial skeletal growth and morphology, we performed craniometric and bone mineral density analyses on skulls from 9-week-old female C57BL/6T germ-free (GF) mice (no microbes), excluded-flora (EF) specific-pathogen-free mice (commensal microbiota), and murine-pathogen-free (MPF) specific-pathogen-free mice (commensal microbiota with SFB). Investigations comparing EF and GF mice revealed that commensal microbiota impacted the size and shape of the craniofacial skeleton. EF versus GF mice exhibited an elongated gross skull length. Cranial bone length analyses normalized to skull length showed that EF versus GF mice had enhanced frontal bone length and reduced cranial base length. The shortened cranial base in EF mice was attributed to decreased presphenoid, basisphenoid, and basioccipital bone lengths. Investigations comparing MPF mice and EF mice demonstrated that commensal gut microbes played a role in craniofacial skeletal morphology. Cranial bone length analyses normalized to skull length showed that MPF versus EF mice had reduced frontal bone length and increased cranial base length. The elongated cranial base in MPF mice was due to enhanced presphenoid bone length. This work, which introduces the commensal microbiota as a contributor to craniofacial skeletal growth, underscores that noninvasive interventions in the gut microbiome could potentially be employed to modify craniofacial skeletal morphology. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Minocycline-induced disruption of the intestinal FXR/FGF15 axis impairs osteogenesis in mice.

Antibiotic-induced shifts in the indigenous gut microbiota influence normal skeletal maturation. Current theory implies that gut microbiota actions on bone occur through a direct gut/bone signaling axis. However, our prior work supports that a gut/liver signaling axis contributes to gut microbiota effects on bone. Our purpose was to investigate the effects of minocycline, a systemic antibiotic treatment for adolescent acne, on pubertal/postpubertal skeletal maturation. Sex-matched specific pathogen-free (SPF) and germ-free (GF) C57BL/6T mice were administered a clinically relevant minocycline dose from age 6-12 weeks. Minocycline caused dysbiotic shifts in the gut bacteriome and impaired skeletal maturation in SPF mice but did not alter the skeletal phenotype in GF mice. Minocycline administration in SPF mice disrupted the intestinal farnesoid X receptor/fibroblast growth factor 15 axis, a gut/liver endocrine axis supporting systemic bile acid homeostasis. Minocycline-treated SPF mice had increased serum conjugated bile acids that were farnesoid X receptor (FXR) antagonists, suppressed osteoblast function, decreased bone mass, and impaired bone microarchitecture and fracture resistance. Stimulating osteoblasts with the serum bile acid profile from minocycline-treated SPF mice recapitulated the suppressed osteogenic phenotype found in vivo, which was mediated through attenuated FXR signaling. This work introduces bile acids as a potentially novel mediator of gut/liver signaling actions contributing to gut microbiota effects on bone.

Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers.

Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.

Nicotinamide induces Fob1-dependent plasmid integration into chromosome XII in Saccharomyces cerevisiae.

In the ribosomal DNA (rDNA) array of Saccharomyces cerevisiae, DNA replication is arrested by the Fob1 protein in a site-specific manner that stimulates homologous recombination. The silent information regulator Sir2, which is loaded at the replication arrest sites by Fob1, suppresses this recombination event. A plasmid containing Fob1-binding sites, when propagated in a yeast strain lacking SIR2 is integrated into the yeast chromosome in a FOB1-dependent manner. We show that addition of nicotinamide (NAM) to the culture medium can stimulate such plasmid integration in the presence of SIR2. Pulsed-field gel electrophoresis analysis showed that plasmid integration occurred into chromosome XII. NAM-induced plasmid integration was dependent on FOB1 and on the homologous recombination gene RAD52. As NAM inhibits several sirtuins, we examined plasmid integration in yeast strains containing deletions of various sirtuin genes and observed that plasmid integration occurred only in the absence of SIR2, but not in the absence of other histone deacetylases. In the absence of PNC1 that metabolizes NAM, a reduced concentration of NAM was required to induce plasmid integration in comparison with that required in wild-type cells. This study suggests that NAD metabolism and intracellular NAM concentrations are important in Fob1-mediated rDNA recombination.

Commensal oral microbiota induces osteoimmunomodulatory effects separate from systemic microbiome in mice.

Commensal microbes critically regulate skeletal homeostasis, yet the impact of specific microbiota communities on osteoimmune response mechanisms is unknown. To discern osteoimmunomodulatory effects imparted by the commensal oral microbiota that are distinct from the systemic microbiota, osteoimmunology studies were performed in both alveolar bone and nonoral skeletal sites of specific pathogen-free (SPF) versus germ-free (GF) mice and SPF mice subjected to saline versus chlorhexidine oral rinses. SPF versus GF mice had reduced cortical/trabecular bone and an enhanced pro-osteoclastic phenotype in alveolar bone. TLR signaling and Th17 cells that have known pro-osteoclastic actions were increased in alveolar BM, but not long BM, of SPF versus GF mice. MHC II antigen presentation genes and activated DCs and CD4+ T cells were elevated in alveolar BM, but not long BM, of SPF versus GF mice. These findings were substantiated by in vitro allostimulation studies demonstrating increased activated DCs derived from alveolar BM, but not long BM, of SPF versus GF mice. Chlorhexidine antiseptic rinse depleted the oral, but not gut, bacteriome in SPF mice. Findings from saline- versus chlorhexidine-treated SPF mice corroborated outcomes from SPF versus GF mice, which reveals that the commensal oral microbiota imparts osteoimmunomodulatory effects separate from the systemic microbiome.

A single strain of Bacteroides fragilis protects gut integrity and reduces GVHD.

Graft-versus-host disease (GVHD) is a pathological process caused by an exaggerated donor lymphocyte response to host antigens after allogeneic hematopoietic cell transplantation (allo-HCT). Donor T cells undergo extensive clonal expansion and differentiation, which culminate in damage to recipient target organs. Damage to the gastrointestinal tract is a main contributor to morbidity and mortality. The loss of diversity among intestinal bacteria caused by pretransplant conditioning regimens leads to an outgrowth of opportunistic pathogens and exacerbated GVHD after allo-HCT. Using murine models of allo-HCT, we found that an increase of Bacteroides in the intestinal microbiota of the recipients was associated with reduced GVHD in mice given fecal microbial transplantation. Administration of Bacteroides fragilis through oral gavage increased gut microbiota diversity and beneficial commensal bacteria and significantly ameliorated acute and chronic GVHD development. Preservation of gut integrity following B. fragilis exposure was likely attributed to increased short chain fatty acids, IL-22, and regulatory T cells, which in turn improved gut tight junction integrity and reduced inflammatory cytokine production of pathogenic T cells. The current study provides a proof of concept that a single strain of commensal bacteria can be a safe and effective means to protect gut integrity and ameliorate GVHD after allo-HCT.

Specific Commensal Bacterium Critically Regulates Gut Microbiota Osteoimmunomodulatory Actions During Normal Postpubertal Skeletal Growth and Maturation.

The commensal gut microbiota critically regulates immunomodulatory processes that influence normal skeletal growth and maturation. However, the influence of specific microbes on commensal gut microbiota osteoimmunoregulatory actions is unknown. We have shown previously that the commensal gut microbiota enhances TH17/IL17A immune response effects in marrow and liver that have procatabolic/antianabolic actions in the skeleton. Segmented filamentous bacteria (SFB), a specific commensal gut bacterium within phylum Firmicutes, potently induces TH17/IL17A-mediated immunity. The study purpose was to delineate the influence of SFB on commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal development. Two murine models were utilized: SFB-monoassociated mice versus germ-free (GF) mice and specific-pathogen-free (SPF) mice +/- SFB. SFB colonization was validated by 16S rDNA analysis, and SFB-induced TH17/IL17A immunity was confirmed by upregulation of Il17a in ileum and IL17A in serum. SFB-colonized mice had an osteopenic trabecular bone phenotype, which was attributed to SFB actions suppressing osteoblastogenesis and enhancing osteoclastogenesis. Intriguingly, SFB-colonized mice had increased expression of proinflammatory chemokines and acute-phase reactants in the liver. Lipocalin-2 (LCN2), an acute-phase reactant and antimicrobial peptide, was substantially elevated in the liver and serum of SFB-colonized mice, which supports the notion that SFB regulation of commensal gut microbiota osteoimmunomodulatory actions are mediated in part through a gut-liver-bone axis. Proinflammatory TH17 and TH1 cells were increased in liver-draining lymph nodes of SFB-colonized mice, which further substantiates that SFB osteoimmune-response effects may be mediated through the liver. SFB-induction of Il17a in the gut and Lcn2 in the liver resulted in increased circulating levels of IL17A and LCN2. Recognizing that IL17A and LCN2 support osteoclastogenesis/suppress osteoblastogenesis, SFB actions impairing postpubertal skeletal development appear to be mediated through immunomodulatory effects in both the gut and liver. This research reveals that specific microbes critically impact commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal growth and maturation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Diagnostic bioluminescent phage for detection of Yersinia pestis.

Yersinia pestis is the etiological agent of the plague. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB ("light")-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage phiA1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28 degrees C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 10(2) cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersinia pseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens.

Susceptibility of gnotobiotic transgenic mice (Tgepsilon26) with combined deficiencies in natural killer cells and T cells to wild-type and hyphal signalling-defective mutants of Candida albicans.

Germfree transgenic epsilon 26 mice (Tgepsilon26), deficient in natural killer cells and T cells, were colonized (alimentary tract) with Candida albicans wild-type or each of two hyphal transcription factor signalling mutant strains (efg1/efg1, efg1/efg1 cph1/cph1). Each Candida strain colonized the alimentary tract, infected keratinized gastric tissues to a similar extent, and induced a granulocyte-dominated inflammatory response in infected tissues. Both wild-type and mutant strains formed hyphae in vivo and were able to elicit an increase in cytokine [tumour necrosis factor alpha, interleukin (IL)-10 and IL-12] and chemokine (KC and macrophage inflammatory protein-2] mRNAs in infected tissues; however, administration of the wild-type strain was lethal for the Tgepsilon26 mice, whereas the mice colonized with the mutant strains survived. Death of the Tgepsilon26-colonized mice appeared to be due to occlusive oesophageal candidiasis, and not to disseminated candidiasis of endogenous origin. In contrast, the mutant strains exhibited a significantly reduced capacity to infect (frequency and severity) oro-oesophageal (tongue and oesophagus) tissues. Therefore, the two hyphal signalling-defective mutants were less able to infect oro-oesophageal tissues and were non-lethal, but retained their ability to colonize the alimentary tract with yeast and hyphae, infect keratinized gastric tissues, and evoke an inflammatory response in orogastric tissues.

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis.

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.

Gut homeostasis and regulatory T cell induction depend on molecular chaperone gp96 in CD11c+ cells.

The intestinal immunity and tolerance are orchestrated by both the innate and the adaptive immune system. Intestinal professional antigen presenting cells (pAPCs) recognize and respond to the gut microbiota through multiple pattern-recognition receptors, including TLRs and NLRs. How gut pAPCs maintain mucosal homeostasis remains incompletely understood. Heat shock protein gp96, also known as grp94, is an essential immune chaperone for TLRs. However, the role of gp96 in regulating CD11c+ APCs in the gut immunity and tolerance is unknown. By a genetic strategy, we report here that selective deletion of gp96 from CD11c+ cells in mice results in alteration of dendritic cell and T cell subsets in the gut as well as loss of antigen-specific regulatory T cell induction in the mesenteric lymph nodes. Strikingly, these conditional gp96-null mice developed spontaneous colitis, had increased levels of systemic and fecal IgA, and were highly susceptible to chemical-induced colitis. Our findings for the first time demonstrate that gp96 is essential for CD11c+ cells to induce regulatory T cells and maintain gut homeostasis, illustrating the importance of protein immune chaperone in safeguarding against immune pathology.

Commensal Gut Microbiota Immunomodulatory Actions in Bone Marrow and Liver have Catabolic Effects on Skeletal Homeostasis in Health.

Despite knowledge the gut microbiota regulates bone mass, mechanisms governing the normal gut microbiota's osteoimmunomodulatory effects on skeletal remodeling and homeostasis are unclear in the healthy adult skeleton. Young adult specific-pathogen-free and germ-free mice were used to delineate the commensal microbiota's immunoregulatory effects on osteoblastogenesis, osteoclastogenesis, marrow T-cell hematopoiesis, and extra-skeletal endocrine organ function. We report the commensal microbiota has anti-anabolic effects suppressing osteoblastogenesis and pro-catabolic effects enhancing osteoclastogenesis, which drive bone loss in health. Suppression of Sp7(Osterix) and Igf1 in bone, and serum IGF1, in specific-pathogen-free mice suggest the commensal microbiota's anti-osteoblastic actions are mediated via local disruption of IGF1-signaling. Differences in the RANKL/OPG Axis in vivo, and RANKL-induced maturation of osteoclast-precursors in vitro, indicate the commensal microbiota induces sustained changes in RANKL-mediated osteoclastogenesis. Candidate mechanisms mediating commensal microbiota's pro-osteoclastic actions include altered marrow effector CD4+T-cells and a novel Gut-Liver-Bone Axis. The previously unidentified Gut-Liver-Bone Axis intriguingly implies the normal gut microbiota's osteoimmunomodulatory actions are partly mediated via immunostimulatory effects in the liver. The molecular underpinnings defining commensal gut microbiota immunomodulatory actions on physiologic bone remodeling are highly relevant in advancing the understanding of normal osteoimmunological processes, having implications for the prevention of skeletal deterioration in health and disease.

CD24 blunts oral squamous cancer development and dampens the functional expansion of myeloid-derived suppressor cells.

CD24 expression has been implicated in the oncogenesis of multiple types of cancer and high tumor expression is considered a poor prognosis factor; however, the role of CD24 in oral cancer progression is unknown. Unlike other cancer types, we found that higher CD24 levels in human oral cancers are correlated to lower clinical stage and better overall survival. We then dissected the role of CD24 and mechanisms in oral cancer pathogenesis in mice using a genetic strategy and demonstrated that CD24 deficiency increased the oral cavity tumor burden in response to the carcinogen 4-nitroquioline 1-oxide (4-NQO). Immune profile analysis showed a significant expansion as well as increased suppressive function of myeloid-derived suppressor cells (MDSCs) in CD24-/- mice, but no apparent impairment in T cells, B cells, or dendritic cells. Further, studies with an orthotopically transplanted syngeneic squamous carcinoma model in the tongue of CD24-/- and CD24+/- mice confirmed the protective roles of CD24 against cancer. Moreover, the difference in tumor growth between CD24-/- and CD24+/- mice was blunted by immunodepletion of MDSCs. We conclude that CD24 expression impedes MDSC expansion and function, and thus slows oral cancer oncogenesis. This study is the first to examine the role of CD24 in a de novo oral cancer model, and it highlights the need to consider the immune regulatory roles of CD24 in the development of CD24-targeted therapy for cancer.

Differential Candida albicans lipase gene expression during alimentary tract colonization and infection.

The human pathogenic fungus Candida albicans, which can reside as a benign commensal of the gut, possesses a large family of lipase encoding genes whose extracellular activity may be important for colonization and subsequent infection. The expression of the C. albicans lipase gene family (LIP1-10) was investigated using a mouse model of mucosal candidiasis during alimentary tract colonization (cecum contents) and orogastric infection. LIPs4-8 were expressed in nearly every sample prepared from the cecum contents and infected mucosal tissues (stomach, hard palate, esophagus and tongue) suggesting a maintenance function for these gene products. In contrast, LIPs1, 3, and 9, which were detected consistently in infected gastric tissues, were essentially undetectable in infected oral tissues. In addition, LIP2 was expressed consistently in cecum contents but was undetectable in infected oral tissues suggesting LIP2 may be important for alimentary tract colonization, but not oral infection. The host responded to a C. albicans infection by significantly increasing expression of the chemokines MIP-2 and KC at the site of infection. Therefore, differential LIP gene expression was observed during colonization, infection and at different infected mucosal sites.

Divergent chemokine, cytokine and beta-defensin responses to gastric candidiasis in immunocompetent C57BL/6 and BALB/c mice.

Previous studies of animal models of candidiasis have produced conflicting results concerning the cytokines and host defence mechanisms that are most relevant for protection against Candida infections. In this study, the host defence mechanisms evoked by two different immunocompetent murine strains following oral colonization with Candida albicans were assessed. beta-Defensin (mBD1, mBD3 and mBD4), chemokine (MIP-2 and KC) and cytokine (TNF-alpha, IFN-gamma, IL-4, IL-10, IL-12 and IL-15) gene expression in germ-free (gf) and C. albicans-infected (gastric) C57BL/6 and BALB/c mice was contrasted. Gf C57BL/6 and BALB/c mice expressed significantly different basal levels of mBD3, mBD4, TNF-alpha and IL-12 in gastric tissues; however, gf C57BL/6 and BALB/c mice were equally susceptible to intestinal colonization with C. albicans and had similar fungal burdens in gastric tissues 4 weeks after oral challenge. C57BL/6 mice responded to colonization and gastric candidiasis with increased expression of mBD1, mBD3, mBD4, TNF-alpha, MIP-2, KC and IL-12. Conversely, a much more specific and attenuated response was observed in Candida-infected gastric tissues from BALB/c mice. Therefore, different strains of mice that were equally susceptible to gastric candidiasis after oral challenge had divergent cytokine, chemokine and beta-defensin responses. This suggests that conflicting data as to the relevance of cytokines and other host factors in murine resistance to candidiasis may be explained, at least in part, by the strain of mouse studied.