Monocyte progenitors give rise to multinucleated giant cells.

The immune response to mycobacteria is characterized by granuloma formation, which features multinucleated giant cells as a unique macrophage type. We previously found that multinucleated giant cells result from Toll-like receptor-induced DNA damage and cell autonomous cell cycle modifications. However, the giant cell progenitor identity remained unclear. Here, we show that the giant cell-forming potential is a particular trait of monocyte progenitors. Common monocyte progenitors potently produce cytokines in response to mycobacteria and their immune-active molecules. In addition, common monocyte progenitors accumulate cholesterol and lipids, which are prerequisites for giant cell transformation. Inducible monocyte progenitors are so far undescribed circulating common monocyte progenitor descendants with high giant cell-forming potential. Monocyte progenitors are induced in mycobacterial infections and localize to granulomas. Accordingly, they exhibit important immunological functions in mycobacterial infections. Moreover, their signature trait of high cholesterol metabolism may be piggy-backed by mycobacteria to create a permissive niche.

Differential effects of commensal bacteria on progenitor cell adhesion, division symmetry and tumorigenesis in the Drosophila intestine.

Microbial factors influence homeostatic and oncogenic growth in the intestinal epithelium. However, we know little about immediate effects of commensal bacteria on stem cell division programs. In this study, we examined the effects of commensal Lactobacillus species on homeostatic and tumorigenic stem cell proliferation in the female Drosophila intestine. We identified Lactobacillus brevis as a potent stimulator of stem cell divisions. In a wild-type midgut, L.brevis activates growth regulatory pathways that drive stem cell divisions. In a Notch-deficient background, L.brevis-mediated proliferation causes rapid expansion of mutant progenitors, leading to accumulation of large, multi-layered tumors throughout the midgut. Mechanistically, we showed that L.brevis disrupts expression and subcellular distribution of progenitor cell integrins, supporting symmetric divisions that expand intestinal stem cell populations. Collectively, our data emphasize the impact of commensal microbes on division and maintenance of the intestinal progenitor compartment.

Epithelial Indoleamine 2,3-Dioxygenase 1 Modulates Aryl Hydrocarbon Receptor and Notch Signaling to Increase Differentiation of Secretory Cells and Alter Mucus-Associated Microbiota.

BACKGROUND & AIMS: Inflammation, injury, and infection up-regulate expression of the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in the intestinal epithelium. We studied the effects of cell-specific IDO1 expression in the epithelium at baseline and during intestinal inflammation in mice. METHODS: We generated transgenic mice that overexpress fluorescence-tagged IDO1 in the intestinal epithelium under control of the villin promoter (IDO1-TG). We generated intestinal epithelial spheroids from mice with full-length Ido1 (controls), disruption of Ido1 (knockout mice), and IDO1-TG and analyzed them for stem cell and differentiation markers by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. Some mice were gavaged with enteropathogenic Escherichia coli (E2348/69) to induce infectious ileitis, and ileum contents were quantified by polymerase chain reaction. Separate sets of mice were given dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid to induce colitis; intestinal tissues were analyzed by histology. We utilized published data sets GSE75214 and GDS2642 of RNA expression data from ilea of healthy individuals undergoing screening colonoscopies (controls) and patients with Crohn's disease. RESULTS: Histologic analysis of small intestine tissues from IDO1-TG mice revealed increases in secretory cells. Enteroids derived from IDO1-TG intestine had increased markers of stem, goblet, Paneth, enteroendocrine, and tuft cells, compared with control enteroids, with a concomitant decrease in markers of absorptive cells. IDO1 interacted non-enzymatically with the aryl hydrocarbon receptor to inhibit activation of NOTCH1. Intestinal mucus layers from IDO1-TG mice were 2-fold thicker than mucus layers from control mice, with increased proportions of Akkermansia muciniphila and Mucispirillum schaedleri. Compared to controls, IDO1-TG mice demonstrated an 85% reduction in ileal bacteria (P = .03) when challenged with enteropathogenic E coli, and were protected from immune infiltration, crypt dropout, and ulcers following administration of dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid. In ilea of Crohn's disease patients, increased expression of IDO1 correlated with increased levels of MUC2, LYZ1, and aryl hydrocarbon receptor, but reduced levels of SLC2A5. CONCLUSIONS: In mice, expression of IDO1 in the intestinal epithelial promotes secretory cell differentiation and mucus production; levels of IDO1 are positively correlated with secretory cell markers in ilea of healthy individuals and Crohn's disease patients. We propose that IDO1 contributes to intestinal homeostasis.

Chronic Chlamydia infection in human organoids increases stemness and promotes age-dependent CpG methylation.

Chronic infections of the fallopian tubes with Chlamydia trachomatis (Ctr) cause scarring and can lead to infertility. Here we use human fallopian tube organoids and genital Ctr serovars D, K and E for long-term in vitro analysis. The epithelial monolayer responds with active expulsion of the bacteria into the lumen and with compensatory cellular proliferation-demonstrating a role of epithelial homeostasis in the defense against this pathogen. In addition, Ctr infection activates LIF signaling, which we find to be an essential regulator of stemness in the organoids. Infected organoids exhibit a less differentiated phenotype with higher stemness potential, as confirmed by increased organoid forming efficiency. Moreover, Ctr increases hypermethylation of DNA, which is an indicator of accelerated molecular aging. Thus, the chronic organoid infection model suggests that Ctr has a long-term impact on the epithelium. These heritable changes might be a contributing factor in the development of tubal pathologies, including the initiation of high grade serous ovarian cancer.

Interaction of bacteria and stem cells in health and disease.

Adult stem and progenitor cells possess unique qualities of proliferative capacity and phenotypic plasticity making their potential interactions with pathogenic and commensal bacteria a significant factor in health and disease. This interaction may result in the hindrance of regenerative capacity and degenerative disease. In other contexts, bacterial-stem cell cross-talk plays an important role in regulating stem cell renewal and maintaining homeostasis. Some stems cells are involved in combating infections and modulating immune responses. The results of these interactions contribute significantly to the outcome of infectious disease. The unique characteristics of stem and progenitor cells also make them attractive targets for bacterial pathogenicity strategies. Several bacterial species have been shown to utilize stem cells as cellular niches or as a means to manipulate host-pathogen interactions. In some cases, bacteria can reprogram end-differentiated tissue cells towards stem-like cells, taking advantage of their unique properties for dissemination and persistence. The ability of bacteria to interfere in stem cell regulatory pathways can also contribute to hyperplastic growth and the development of cancer. In this review, we present current knowledge on the diverse interactions between bacteria and stem cells highlighting the consequences for health and disease.

Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice.

Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective.

Epithelial IL-23R Signaling Licenses Protective IL-22 Responses in Intestinal Inflammation.

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.

The role of primary infection of Schwann cells in the aetiology of infective inflammatory neuropathies.

Numerous different pathogens are responsible for infective peripheral neuropathies and this is generally the result of the indirect effects of pathogen infection, namely anti pathogen antibodies cross reacting with epitopes on peripheral nerve, auto reactive T cells attacking myelin, circulating immune complexes and complement fixation. Primary infection of Schwann cells (SC) associated with peripheral nerve inflammation is rare requiring pathogens to cross the Blood Peripheral Nerve Barrier (BPNB) evade anti-pathogen innate immune pathways and invade the SC. Spirochetes Borrelia bourgdorferi and Trepomema pallidum are highly invasive, express surface lipo proteins, but despite this SC are rarely infected. However, Trypanosoma cruzi (Chaga's disease) and Mycobacterium leprae. Leprosy are two important causes of peripheral nerve infection and both demonstrate primary infection of SC. This is due to two novel strategies; T. cruzi express a trans-silalidase that mimics host neurotrophic factors and infects SC via tyrosine kinase receptors. M. leprae demonstrates multi receptor SC tropism and subsequent infection promotes nuclear reprogramming and dedifferentiation of host SC into progenitor stem like cells (pSLC) that are vulnerable to M. leprae infection. These two novel pathogen evasion strategies, involving stem cells and receptor mimicry, provide potential therapeutic targets relevant to the prevention of peripheral nerve inflammation by inhibiting primary SC infection.

Intestinal stem cells and intestinal homeostasis in health and in inflammation: A review.

BACKGROUND: The human intestine is a complex group of organs, highly specialized in processing food and providing nutrients to the body. It is under constant threat from microbials and toxins and has therefore developed a number of protective mechanisms. One important mechanism is the constant shedding of epithelial cells into the lumen; another is the production and maintenance of a double-layered mucous boundary in which there is continuous sampling of the luminal microbiota and a persistent presence of antimicrobial enzymes. However, the gut needs commensal bacteria to effectively break down food into absorbable nutrients, which necessitates constant communication between the luminal bacteria and the intestinal immune cells in homeostasis. Disruption of homeostasis, for whatever reason, will give rise to (chronic) inflammation. DISCUSSION: Both medical and surgical management of this disruption is discussed.

Analysis of Macrophage-Induced Salmonella Persisters.

A small subpopulation of non-replicating, multidrug-tolerant bacteria is present within clonal populations of many bacterial species. Known as persisters, these bacteria are probably the cause of relapsing infections such as typhoid fever. Formation of non-growing Salmonella persisters is stimulated by macrophage phagocytosis. This chapter outlines methods to identify and study persisters resulting from interactions between bacterial pathogens and their hosts. We use their antibiotic tolerance for isolation and enumeration and developed a method to study the heterogeneity of growth within clonal populations through single-cell analysis.

Oral Mucosal Lamina Propria-Progenitor Cells Exert Antibacterial Properties via the Secretion of Osteoprotegerin and Haptoglobin.

UNLABELLED: The oral cavity possesses a diverse microflora, yet recurrent infections within healthy individuals are rare. Wound healing within the buccal mucosa is preferential, potentially because of the presence of oral mucosal lamina propria-progenitor cells (OMLP-PCs). In addition to their multipotency, OMLP-PCs demonstrate potent immunosuppressive properties. The present study investigated whether OMLP-PCs possess antibacterial properties, directly interacting with microorganisms and contributing to the maintenance of a balanced oral microflora. Gram-positive and -negative bacteria were cocultured with OMLP-PCs, buccal mucosal fibroblasts, or their respective conditioned media (CM). Bacterial growth was significantly inhibited when cocultured with OMLP-PCs or their CM. No antibacterial activity was apparent within the fibroblasts. Analysis of the OMLP-PC CM indicated constitutive secretion of osteoprotegerin (OPG) and haptoglobin (Hp). Exposure of the bacteria to OPG or Hp demonstrated their differential antibacterial properties, with neutralization/blocking studies confirming that the growth of Gram-positive bacteria was partially restored by neutralizing OPG within OMLP-PC CM; blocking Hp restored the growth of Gram-negative bacteria. The present study demonstrates, for the first time, the broad-spectrum antibacterial properties of OMLP-PCs. We report the direct and constitutive antibacterial nature of OMLP-PCs, with retention of this effect within the CM suggesting a role for soluble factors such as OPG and Hp. Knowledge of the immunomodulatory and antibacterial properties of these cells could potentially be exploited in the development of novel cell- or soluble factor-based therapeutics for the treatment of infectious diseases such as pneumonia or ailments such as chronic nonhealing wounds. SIGNIFICANCE: Oral mucosal lamina propria-progenitor cells (OMLP-PCs) are a cell source with known immunomodulatory properties. The present report demonstrates the novel finding that OMLP-PCs possess potent antibacterial properties, halting the growth of Gram-positive and -negative bacteria through the secretion of soluble factors. OMLP-PCs constitutively secrete osteoprotegerin (OPG) and haptoglobin (Hp) at levels high enough to exert antibacterial action. OPG, a glycoprotein not previously known to be antibacterial, can suppress Gram-positive bacterial growth. Hp is only active against Gram-negative microorganisms. These findings indicate that OMLP-PCs could offer great potential in the development of novel cell- or soluble factor-based therapies for the treatment of infectious illness, such as bacterial pneumonia, through systemic infusion and of chronic wounds through local administration.

Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands.

BACKGROUND & AIMS: Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology. METHODS: We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. RESULTS: H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. CONCLUSIONS: H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.

Risk-assessment-based approach to patients exposed to endoscopes contaminated with Pseudomonas spp.

Patients exposed to bronchoscopes contaminated with Pseudomonas aeruginosa are at increased risk of pseudomonal infection. The optimal methods for management and mitigation of risk following exposure are controversial. This article describes a two-phase risk assessment following pseudomonal contamination of a family of 75 endoscopes, detected through routine surveillance and attributed to one endoscope washer-disinfector. An initial risk assessment identified 18 endoscopes as high risk, based on the presence of lumens used for irrigation or biopsy. Exposure was communicated to the patients' clinical teams and a further clinical risk assessment of the exposed patients was performed. No patients developed complications due to pseudomonal infection.

In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection.

BACKGROUND & AIMS: We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. METHODS: We generated organoids from surgical samples of human gastric corpus. Culture conditions were developed based on those for the mouse gastric and human intestinal systems. We used microinjection to infect the organoids with H pylori. Epithelial responses were measured using microarray and quantitative polymerase chain reaction analyses. RESULTS: Human gastric cells were expanded indefinitely in 3-dimensional cultures. We cultured cells from healthy gastric tissues, single-sorted stem cells, or tumor tissues. Organoids maintained many characteristics of their respective tissues based on their histology, expression of markers, and euploidy. Organoids from healthy tissue expressed markers of 4 lineages of the stomach and self-organized into gland and pit domains. They could be directed to specifically express either lineages of the gastric gland, or the gastric pit, by addition of nicotinamide and withdrawal of WNT. Although gastric pit lineages had only marginal reactions to bacterial infection, gastric gland lineages mounted a strong inflammatory response. CONCLUSIONS: We developed a system to culture human gastric organoids. This system can be used to study H pylori infection and other gastric pathologies.

Interactions of anaerobic bacteria with dental stem cells: an in vitro study.

BACKGROUND: In patients with periodontitis, it is highly likely that local (progenitor) cells encounter pathogenic bacteria. The purpose of this in vitro study was to elucidate how human dental follicle stem cells (hDFSC) react towards a direct challenge with anaerobic periodontal pathogens under their natural oxygen-free atmosphere. HDFSC were compared to human bone marrow mesenchymal stem cells (hBMSC) and differentiated primary human gingival fibroblasts (hGiF), as well as permanent gingival carcinoma cells (Ca9-22). METHODOLOGY/PRINCIPAL FINDINGS: The different cell types were investigated in a co-culture system with Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). The viability of the cells and pathogens under anaerobic conditions, as well as interactions in terms of adherence and internalization, were examined. Additionally, the release of pro-inflammatory interleukin-8 (IL-8) and anti-inflammatory interleukin-10 (IL-10) was quantified via enzyme-linked immunosorbent assay. The bacteria adhered less efficiently to hDFSC compared to Ca9-22 (P. gingivalis: 0.18% adherence to hDFSC; 3.1% adherence to Ca9-22). Similar results were observed for host cell internalization (F. nucleatum: 0.002% internalization into hDFSC; 0.09% internalization into Ca9-22). Statistically significantly less IL-8 was secreted from hDFSC after stimulation with F. nucleatum and P. gingivalis in comparison with hGiF (F. nucleatum: 2080.0 pg/ml--hGiF; 19.7 pg/ml--hDFSC). The IL-10 response of the differentiated cells was found to be low in relation to their pro-inflammatory IL-8 response. CONCLUSIONS/SIGNIFICANCE: The results indicate that dental stem cells are less prone to interactions with pathogenic bacteria than differentiated cells in an anaerobic environment. Moreover, during bacterial challenge, the stem cell immune response seems to be more towards an anti-inflammatory reaction. For a potential future therapeutic use of hDFSC, these findings support the idea of a save application.

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function.

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4(+) T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼ 30-fold increase in Sca-1(hi) progenitors and a corresponding loss of Sca-1(lo/int) subsets. Most strikingly, the capacity of donor Sca-1(hi) cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1(hi) c-kit(int) cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.

Role of docosahexaenoic acid treatment in improving liver histology in pediatric nonalcoholic fatty liver disease.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is one of the most important causes of liver-related morbidity and mortality in children. Recently, we have reported the effects of docosahexaenoic acid (DHA), the major dietary long-chain polyunsaturated fatty acids, in children with NAFLD. DHA exerts a potent anti-inflammatory activity through the G protein-coupled receptor (GPR)120. Our aim was to investigate in pediatric NAFLD the mechanisms underlying the effects of DHA administration on histo-pathological aspects, GPR120 expression, hepatic progenitor cell activation and macrophage pool. PATIENTS AND METHODS: 20 children with untreated NAFLD were included. Children were treated with DHA for 18 months. Liver biopsies before and after the treatment were analyzed. Hepatic progenitor cell activation, macrophage pool and GPR120 expression were evaluated and correlated with clinical and histo-pathological parameters. RESULTS: GPR120 was expressed by hepatocytes, liver macrophages, and hepatic progenitor cells. After DHA treatment, the following modifications were present: i) the improvement of histo-pathological parameters such as NAFLD activity score, ballooning, and steatosis; ii) the reduction of hepatic progenitor cell activation in correlation with histo-pathological parameters; iii) the reduction of the number of inflammatory macrophages; iv) the increase of GPR120 expression in hepatocytes; v) the reduction of serine-311-phosphorylated nuclear factor kappa B (NF-κB) nuclear translocation in hepatocytes and macrophages in correlation with serum inflammatory cytokines. CONCLUSIONS: DHA could modulate hepatic progenitor cell activation, hepatocyte survival and macrophage polarization through the interaction with GPR120 and NF-κB repression. In this scenario, the modulation of GPR120 exploits a novel crucial role in the regulation of the cell-to-cell cross-talk that drives inflammatory response, hepatic progenitor cell activation and hepatocyte survival.

Ras-oncogenic Drosophila hindgut but not midgut cells use an inflammation-like program to disseminate to distant sites.

The gastrointestinal tract is habitable by a variety of microorganisms and it is often a tissue inflicted by inflammation. Much discussion is raised in recent years about the role of microbiota in intestinal inflammation, but their role in intestinal cancer remains unclear. Here we discuss and extent our work on Drosophila melanogaster models of tumorigenesis and tumor cell invasion upon intestinal infection. In Drosophila midgut bacteria that cause enterocyte damage induce intestinal stem cell proliferation, which is diverted toward aberrant stem cell expansion upon oncogene expression to induce dysplastic tumors. In the hindgut though, oncogenes synergize with the innate immune response-not the bacterially mediated damage-to induce tumor cell invasion and dissemination to distant sites. Interestingly, our novel gene expression analysis of Drosophila hemocyte-like cells suggests commonalities with oncogenic hindgut cells in the innate immune response and the expression of matrix metalloproteinase 1 in response to bacterial infection.