Exopolysaccharide-Treated Dendritic Cells Effectively Ameliorate Acute Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is a primary and often lethal complication of allogenic hematopoietic stem cell transplantation (HSCT). Prophylactic regimens for GVHD are given as standard pretransplantation therapy; however, up to 50% of these patients develop acute GVHD (aGVHD) and require additional immunosuppressive intervention. Using a mouse GVHD model, we previously showed that injecting mice with exopolysaccharide (EPS) from Bacillus subtilis prior to GVHD induction significantly increased 80-day survival after transplantation of complete allogeneic major histocompatibility complex-mismatched cells. To ask whether EPS might also inhibit GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of A2neg human peripheral blood mononuclear cells (PBMCs). Because we could not inject human donors with EPS, we transferred EPS-pretreated dendritic cells (DCs) to inhibit aGVHD. We derived these DCs from CD34+ human cord blood cells, treated them with EPS, and then injected them together with PBMCs into the NSG-HLA-A2 mice. We found that all mice that received untreated DCs were dead by day 35, whereas 25% of mice receiving EPS-treated DCs (EPS-DCs) survived. This DC cell therapy could be readily translatable to humans, because we can generate large numbers of human EPS-DCs and use them as an "off the shelf" treatment for patients undergoing HSCT.

The effect of an exopolysaccharide probiotic molecule from Bacillus subtilis on breast cancer cells.

Introduction: Many well-known risk factors for breast cancer are associated with dysbiosis (an aberrant microbiome). However, how bacterial products modulate cancer are poorly understood. In this study, we investigated the effect of an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis on breast cancer phenotypes. Although B. subtilis is commonly included in probiotic preparations and its EPS protects against inflammatory diseases, it was virtually unknown whether B. subtilis-derived EPS affects cancer. Methods: This work investigated effects of EPS on phenotypes of breast cancer cells as a cancer model. The phenotypes included proliferation, mammosphere formation, cell migration, and tumor growth in two immune compromised mouse models. RNA sequencing was performed on RNA from four breast cancer cells treated with PBS or EPS. IKKß or STAT1 signaling was assessed using pharmacologic or RNAi-mediated knock down approaches. Results: Short-term treatment with EPS inhibited proliferation of certain breast cancer cells (T47D, MDA-MB-468, HCC1428, MDA-MB-453) while having little effect on others (MCF-7, MDA-MB-231, BT549, ZR-75-30). EPS induced G1/G0 cell cycle arrest of T47D cells while increasing apoptosis of MDA-MB-468 cells. EPS also enhanced aggressive phenotypes in T47D cells including cell migration and cancer stem cell survival. Long-term treatment with EPS (months) led to resistance in vitro and promoted tumor growth in immunocompromised mice. RNA-sequence analysis showed that EPS increased expression of pro-inflammatory pathways including STAT1 and NF-κB. IKKß and/or STAT1 signaling was necessary for EPS to modulate phenotypes of EPS sensitive breast cancer cells. Discussion: These results demonstrate a multifaceted role for an EPS molecule secreted by the probiotic bacterium B. subtilis on breast cancer cell phenotypes. These results warrant future studies in immune competent mice and different cancer models to fully understand potential benefits and/or side effects of long-term use of probiotics.

Mechanism of TLR4-Mediated Anti-Inflammatory Response Induced by Exopolysaccharide from the Probiotic Bacillus subtilis.

Intestinal inflammatory diseases affect millions of people worldwide, and one class of drugs showing promise toward treatment of several inflammatory diseases is probiotics. Numerous studies have been performed using probiotics to prevent and treat intestinal inflammatory diseases. Most of these studies used intact bacteria, and neither the active molecule nor the molecular mechanisms by which they affect immune responses are known. We have shown that the probiotic Bacillus subtilis is anti-inflammatory and can protect mice from acute colitis induced by the enteric pathogen Citrobacter rodentium. We identified and purified the active molecule, exopolysaccharide (EPS), and showed that it protects mice from C. rodentium-induced colitis by inducing anti-inflammatory M2 macrophages or inhibitory dendritic cells (DCs), both of which inhibit excessive T cell responses. We showed previously that EPS affects macrophages and DCs in a TLR4-dependent manner, and in the current study we asked how EPS induces these anti-inflammatory cells and how they function to inhibit T cells. By investigating the signaling downstream of TLR4 that leads to acquisition of inhibitory properties of macrophages and DCs, we found that EPS induces expression of the inhibitory molecule IDO in bone marrow-derived DCs, and that inhibition of T cell proliferation by IDO-expressing bone marrow-derived DCs utilizes the kynurenine/aryl hydrocarbon receptor circuit. Furthermore, unlike LPS, EPS does not induce inflammatory cytokines upon injection in vivo, directly demonstrating different outcomes induced by two different TLR4 agonists.

Death of tonsillar B cells by NETosis.

Regulating B cell death is essential for generating antibodies and maintaining immune tolerance. B cells can die by apoptosis, and we report that human tonsil B cells, but not peripheral blood B cells also die by NETosis. This cell death is density-dependent, characterized by the loss of cell and nuclear membrane integrity, release of reactive oxygen species, and chromatin decondensation. Tonsil B cells secrete high levels of TNF, and inhibiting TNF prevented chromatin decondensation. By in situ fluorescence microscopy, B cell NETosis, as identified by the hyper citrullination of Histone-3, was localized to the light zone (LZ) of germinal centers in normal tonsil and overlapped with the B cell marker CD19/IgM. We propose a model in which stimulation of B cells in the LZ induces NETosis, driven in part by TNF. We also provide evidence that NETosis of tonsil B cells may be inhibited by an unidentified factor in tonsil. The results describe a previously unidentified form of B cell death and suggest a new mechanism to maintain B cell homeostasis during immune responses.

Amelioration of Graft-versus-Host Disease by Exopolysaccharide from a Commensal Bacterium.

Acute graft-versus-host disease (aGvHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation, and although prophylactic regimens are given as standard pretransplantation therapy, up to 60% of these patients develop aGvHD, and require additional immunosuppressive intervention. We treated mice with a purified probiotic molecule, exopolysaccharide (EPS) from Bacillus subtilis, shortly before and after induction of aGvHD and found that, whereas only 10% of control mice survived to day 80, 70% of EPS-treated mice survived to 80 d. EPS treatment of donor-only mice resulted in ∼60% survival. Using a biosensor mouse model to assess inflammation in live mice during aGvHD, we found that EPS prevented the activation of alloreactive donor T cells. In vitro, EPS did not affect T cells directly but, instead, induced bone marrow-derived dendritic cells (BMDCs) that displayed characteristics of inhibitory dendritic cells (DCs). Development of these BMDCs required TLR4 signaling through both MyD88 and TRIF pathways. Using BMDCs derived from IDO knockout mice, we showed that T cell inhibition by EPS-treated BMDCs was mediated through the suppressive effects of IDO. These studies describe a bacterial molecule that modulates immune responses by inducing inhibitory DCs in a TLR4-dependent manner, and these cells have the capacity to inhibit T cell activation through IDO. We suggest that EPS or EPS-treated DCs can serve as novel agents for preventing aGvHD.

Human CD36hi monocytes induce Foxp3+ CD25+ T cells with regulatory functions from CD4 and CD8 subsets.

The fetal and neonatal immune systems are uniquely poised to generate tolerance to self, maternal and environmental antigens encountered in the womb and shortly after birth. However, the tolerogenic nature of fetal and neonatal immunity can be detrimental in the context of pathogens, leading to overwhelming bacterial infections or chronic viral infections. A variety of mechanisms contribute to fetal and neonatal tolerance, including a propensity to generate Foxp3+ regulatory T cells (Treg cells). However, the mechanism(s) of fetal Foxp3+ T-cell differentiation, the specific antigen-presenting cells required and factors that inhibit Treg generation after the neonatal period are poorly understood. Here, we demonstrate that a subset of CD14+ monocytes expressing the scavenger molecule, CD36, can generate CD4+ and CD8+ T cells that coexpress Foxp3 and T-bet from both umbilical cord blood. These Foxp3+ T-bet+ T cells potently suppress T-cell proliferation and ameliorate xenogeneic graft-versus-host disease. CD14+  CD36+ monocytes provide known Treg-inducing signals: membrane-bound transforming growth factor-beta and retinoic acid. Unexpectedly, adult peripheral blood monocytes are also capable of inducing Foxp3+ T cells from both cord blood and adult peripheral naïve T cells. The induction of Foxp3+ T cells in umbilical cord blood by monocytes was inhibited by the lymphoid fraction of adult peripheral blood cells. These studies highlight a novel immunoregulatory role of monocytes and suggest that antigen presentation by CD36hi monocytes may contribute to the peripheral development of Foxp3+ T-bet+ T cells with regulatory functions in both neonates and adults.

Suppression of Staphylococcus aureus Superantigen-Independent Interferon Gamma Response by a Probiotic Polysaccharide.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes a variety of diseases. Bloodstream infection is the most severe, with mortality rates reaching 20 to 50%. Exopolysaccharide (EPS) from the probiotic Bacillus subtilis reduces bacterial burden and inflammation during S. aureus bloodstream infection in mice. Protection is due, in part, to hybrid macrophages that restrict S. aureus growth through reactive oxygen species and to limiting superantigen-induced T cell activation and interferon gamma (IFN-γ) production during infection. A decrease in IFN-γ production was observed within 24 h after infection, and here, we investigated how EPS abrogates its production. We discovered that S. aureus uses a rapid, superantigen-independent mechanism to induce host IFN-γ and that this is mediated by interleukin-12 (IL-12) activation of NK cells. Furthermore, we found that EPS limits IFN-γ production by modulating host immunity in a Toll-like receptor 4 (TLR4)-dependent manner, a signaling pathway that is required for EPS-mediated protection from S. aureus infection in vivo We conclude that EPS protects hosts from acute bloodstream S. aureus infection not only by inducing macrophages that restrict S. aureus growth and inhibit superantigen-activated T cells but also by limiting NK cell production of IFN-γ after S. aureus infection in a TLR4-dependent manner.

Antibiotics Drive Microbial Imbalance and Vitiligo Development in Mice.

Vitiligo is impacted by environmental triggers. We studied the contribution of the microbiome in FH mice, in which depigmentation is mediated by tyrosinase-reactive T cells. The mice received oral antibiotics and were monitored for depigmentation. The microbiome was studied in fecal and skin samples using 16S rRNA analysis. The resulting T-cell distributions were evaluated. In untreated mice, pigment loss did not expand to the pelage, whereas mice in the ampicillin group were approximately 1/3 depigmented at 30 weeks. In contrast to models of autoimmunity that are less dependent on IFN-γ, ampicillin but not neomycin treatment correlated with accelerated disease and reduced bacteria in the fecal pellets. Modified cytokine patterns in the tissue and serum suggest a response that transcends the gut. Ampicillin-induced depigmentation was accompanied by gut but not skin dysbiosis, and reduced T cell numbers in both sites. Neomycin induced a redistribution of gut T cells and an accumulation of skin regulatory T cells. This treatment spurred a Bacteroides-dominated population of fecal bacteria. Reduced diversity is prominent particularly after ampicillin treatment, when the gut is dominated by Pseudomonas species. In line with current concepts relating the microbiome and the immune system, we predict that dietary measures might promote skin health and delay vitiligo onset.

A Caspase-1 Biosensor to Monitor the Progression of Inflammation In Vivo.

Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro-IL-1ß and pro-IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow-derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.

Author Correction: The wide utility of rabbits as models of human diseases.

This article was originally published under a CC BY-NC-SA License, but has now been made available under a CC BY 4.0 License.

Probiotic Exopolysaccharide Protects against Systemic Staphylococcus aureus Infection, Inducing Dual-Functioning Macrophages That Restrict Bacterial Growth and Limit Inflammation.

Staphylococcus aureus causes severe systemic infection with high mortality rates. We previously identified exopolysaccharide (EPS) from a probiotic, Bacillus subtilis, that induces anti-inflammatory macrophages with an M2 phenotype and protects mice from Citrobacter rodentium-induced colitis. We tested if EPS could protect from systemic infection induced by S. aureus and found that EPS-treated mice had enhanced survival as well as reduced weight loss, systemic inflammation, and bacterial burden. While macrophages from EPS-treated mice display an M2 phenotype, they also restrict growth of internalized S. aureus through reactive oxygen species (ROS), reminiscent of proinflammatory phagocytes. These EPS-induced macrophages also limit T cell activation by S. aureus superantigens, and EPS abrogates systemic induction of gamma interferon after infection. We conclude that B. subtilis EPS is an immunomodulatory agent that induces hybrid macrophages that bolster antibacterial immunity and simultaneously limit inflammation, reducing disease burden and promoting host survival.

Identification of a new European rabbit IgA with a serine-rich hinge region.

In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of IGHVa1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed.

The wide utility of rabbits as models of human diseases.

Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur's rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.

The remnant of the European rabbit (Oryctolagus cuniculus) IgD gene.

Although IgD first appeared, along with IgM, in the cartilaginous fishes and has been retained throughout subsequent vertebrate evolution, it has been lost in a diverse group of vertebrate species. We previously showed that, unlike vertebrates that express IgD, the rabbit lacks an IgD (Cδ) gene within 13.5 kb downstream of the IgM gene. We report here that, by conducting BLAST searches of rabbit Ig heavy chain genomic DNA with known mammalian IgD exons, we identified the remnant of the rabbit Cδ gene approximately 21 kb downstream of the IgM gene. The remnant Cδ locus lacks the δCH1 and hinge exons, but contains truncated δCH2 and δCH3 exons, as well as largely intact, but non-functional, secretory and transmembrane exons. In addition, we report that the Cδ gene probably became non-functional in leporids at least prior to the divergence of rabbits and hares ~12 million years ago.

Inflammatory Changes in Bone Marrow Microenvironment Associated with Declining B Lymphopoiesis.

B lymphopoiesis arrests precipitously in rabbits such that by 2-4 mo of age, before sexual maturity, little to no B lymphopoiesis occurs in the bone marrow (BM). Previously, we showed that in mice, adipocytes inhibit B lymphopoiesis in vitro by inducing inflammatory myeloid cells, which produce IL-1ß. In this study, we characterized rabbit BM after the arrest of B lymphopoiesis and found a dramatic increase in fat, increased CD11b+ myeloid cells, and upregulated expression of the inflammatory molecules, IL-1ß and S100A9, by the myeloid cells. We added BM fat, CD11b+ myeloid cells, and recombinant S100A9 to B lymphopoiesis cultures and found that they inhibited B lymphopoiesis and enhanced myelopoiesis. Unlike IL-1ß, which inhibits B lymphopoiesis by acting on early lymphoid progenitors, S100A9 inhibits B lymphopoiesis by acting on myeloid cells and promoting the release of inflammatory molecules, including IL-1ß. Many molecules produced by adipocytes activate the NLRP3 inflammasome, and the NLRP3 inhibitor, glibenclamide, restored B lymphopoiesis and minimized induction of myeloid cells induced by adipocyte-conditioned medium in vitro. We suggest that fat provides an inflammatory microenvironment in the BM and promotes/activates myeloid cells to produce inflammatory molecules such as IL-1ß and S100A9, which negatively regulate B lymphopoiesis.

Exopolysaccharide from Bacillus subtilis Induces Anti-Inflammatory M2 Macrophages That Prevent T Cell-Mediated Disease.

Commensal bacteria contribute to immune homeostasis in the gastrointestinal tract; however, the underlying mechanisms for this are not well understood. A single dose of exopolysaccharide (EPS) from the probiotic spore-forming bacterium Bacillus subtilis protects mice from acute colitis induced by the enteric pathogen Citrobacter rodentium Adoptive transfer of macrophage-rich peritoneal cells from EPS-treated mice confers protection from disease to recipient mice. In vivo, EPS induces development of anti-inflammatory M2 macrophages in a TLR4-dependent manner, and these cells inhibit T cell activation in vitro and in C. rodentium-infected mice. In vitro, M2 macrophages inhibit CD4+ and CD8+ T cells. The inhibition of CD4+ T cells is dependent on TGF-ß, whereas inhibition of CD8+ T cells is dependent on TGF-ß and PD-L1. We suggest that administration of B. subtilis EPS can be used to broadly inhibit T cell activation and, thus, control T cell-mediated immune responses in numerous inflammatory diseases.

Bone marrow fat and the decline of B lymphopoiesis in rabbits.

B lymphopoiesis is necessary to generate a diverse pool of naïve B cells that are able to respond to a broad spectrum of antigens during immune responses to pathogens and to vaccination. Rabbits have been utilized for many years to generate high affinity monoclonal and polyclonal antibodies. Specific antibodies generated in rabbits have greatly advanced scientific discoveries, but the unique qualities of rabbit B cell development have been underappreciated. Unlike in humans and mice, where B lymphopoiesis declines in mid to late life, B lymphopoiesis in rabbits arrests early in life, between 2 and 4 months of age. This review focuses on the early loss of B cell development in rabbits and the contribution of the bone marrow microenvironment to this process. We also propose directions for future research in this area, and discuss how the rabbit can be used as a model to understand the decline of B lymphopoiesis that occurs in humans late in life. Such studies will be important for developing therapeutics targeted to prevent and/or reverse declining B lymphopoiesis in the elderly, as well as boosting immunity and antibody responses after infection or vaccination.

Diversification of the Primary Antibody Repertoire by AID-Mediated Gene Conversion.

Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.