Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

A randomized trial of a lab-embedded discourse intervention to improve research ethics.

We report a randomized trial of a research ethics training intervention designed to enhance ethics communication in university science and engineering laboratories, focusing specifically on authorship and data management. The intervention is a project-based research ethics curriculum that was designed to enhance the ability of science and engineering research laboratory members to engage in reason giving and interpersonal communication necessary for ethical practice. The randomized trial was fielded in active faculty-led laboratories at two US research-intensive institutions. Here, we show that laboratory members perceived improvements in the quality of discourse on research ethics within their laboratories and enhanced awareness of the relevance and reasons for that discourse for their work as measured by a survey administered over 4 mo after the intervention. This training represents a paradigm shift compared with more typical module-based or classroom ethics instruction that is divorced from the everyday workflow and practices within laboratories and is designed to cultivate a campus culture of ethical science and engineering research in the very work settings where laboratory members interact.

Chronic Inflammation in Mucosal Tissues: Barrier Integrity, Inducible Lymphoid Tissues, and Immune Surveillance.

An interesting phenomenon of chronic inflammation is that the associated cytokines can simultaneously promote inflammatory cell recruitment and tissue pathology as well as tissue regeneration and development of inducible organized lymphoid tissues (tertiary lymphoid organs or TLO), demonstrating the remarkable dynamics of the immune interactions with host tissues. In mucosal tissues, chronic immune-mediated inflammation can present a mixed inflammatory pathology including neutrophil infiltrates along with the lymphocytic aggregates. The factors driving this pattern may involve effects on barrier function as well as inducible mechanisms associated with immune surveillance. The relative contribution of these factors may be important in determining the outcome, from resolution to inflammatory stalemate to progressive tissue pathology and destruction. Here, we focus on the specific impact of cytokine-driven inducible lymphoid cells and tissues on immune surveillance at mucosal surfaces, including the induction of epithelial M cells. We propose a model of chronic intestinal inflammation to assess the relative contributions of mucosal barrier integrity, M cell transcytosis of luminal microbes, and inducible lymphoid tissues.

Vigilance or Subversion? Constitutive and Inducible M Cells in Mucosal Tissues.

Microfold (M) cells are epithelial cells present in mucosal tissues and specialized for the capture of luminal microparticles and their delivery to underlying immune cells; thus, they are crucial participants in mucosal immune surveillance. Multiple phenotypic subsets of M cells have now been described, all sharing a unique apical morphology that provides clues to their ability to capture microbial particles. The existence of diverse M cell phenotypes, especially inflammation-inducible M cells, provides an intriguing puzzle: some variants may augment luminal surveillance to boost mucosal immunity, while others may promote microbial access to tissues. Here, I consider the unique induction requirements of each M cell subset and functional differences, highlighting the potentially distinct consequences in mucosal immunity.

Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

Acute Immune Response of Micro- and Nanosized Erythrocyte-Derived Optical Particles in Healthy Mice.

Erythrocyte-derived particles activated by near-infrared (NIR) light present a platform for various phototheranostic applications. We have engineered such a platform with indocyanine green as the NIR-activated agent. A particular feature of these particles is that their diameters can be tuned from micro- to nanoscale, providing a potential capability for broad clinical utility ranging from vascular to cancer-related applications. An important issue related to clinical translation of these particles is their immunogenic effects. Herein, we have evaluated the early-induced innate immune response of these particles in healthy Swiss Webster mice following tail vein injection by measurements of specific cytokines in blood serum, the liver, and the spleen following euthanasia. In particular, we have investigated the effects of particle size and relative dose, time-dependent cytokine response for up to 6 h postinjection, functionalization of the nanosized particles with folate or Herceptin, and dual injections of the particles 1 week apart. Mean concentrations of interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1 in response to injection of microsized particles at the investigated relative doses were significantly lower than the corresponding mean concentrations induced by lipopolysaccharide (positive control) at 2 h. All investigated doses of the nanosized particles induced significantly higher concentrations of MCP-1 in the liver and the spleen as compared to phosphate buffer saline (PBS) (negative control) at 2 h. In response to micro- and nanosized particles at the highest investigated dose, there were significantly higher levels of TNF-α in blood serum at 2 and 6 h postinjection as compared to the levels associated with PBS treatment at these times. Whereas the mean concentration of TNF-α in the liver significantly increased between 2 and 6 h postinjection in response to the injection of the microsized particles, it was significantly reduced during this time interval in response to the injection of the nanosized particles. In general, functionalization of the nanosized particles was associated with a reduction of IL-6 and MCP-1 in blood serum, the liver, and the spleen, and TNF-α in blood serum. With the exception of IL-10 in the spleen in response to nanosized particles, the second injection of micro- or nanosized particles did not lead to significantly higher concentrations of other cytokines at the investigated dose as compared to a single injection.

Crosslinked flagella as a stabilized vaccine adjuvant scaffold.

BACKGROUND: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. RESULTS: Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. CONCLUSIONS: We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

Induction of Colonic M Cells during Intestinal Inflammation.

Intestinal M (microfold) cells are specialized epithelial cells overlying lymphoid tissues in the small intestine. Unlike common enterocytes, M cells lack an organized apical brush border, and are able to transcytose microparticles across the mucosal barrier to underlying antigen-presenting cells. We found that in both the dextran sodium sulfate and Citrobacter rodentium models of colitis, significantly increased numbers of Peyer's patch (PP) phenotype M cells were induced at the peak of inflammation in colonic epithelium, often accompanied by loosely organized lamina propria infiltrates. PP type M cells are thought to be dependent on cytokines, including tumor necrosis factor (TNF)-α and receptor activator of nuclear factor kappa-B ligand; these cytokines were also found to be induced in the inflamed tissues. The induction of M cells was abrogated by anti-TNF-α blockade, suggesting that anti-TNF-α therapies may have similar effects in clinical settings, although the functional consequences are not clear. Our results suggest that inflammatory cytokine-induced PP type M cells may be a useful correlate of chronic intestinal inflammation.

Molecular Mechanism of Biased Ligand Conformational Changes in CC Chemokine Receptor 7.

Biased ligand binding to G protein-coupled receptors enables functional selectivity of intracellular effectors to mediate cellular function. Despite the significant advances made in characterizing the conformational states (transmembrane helical arrangements) capable of discriminating between G protein and arrestin binding, the role of the ligand in stabilizing such conformations remains unclear. To address this issue, we simulate microsecond dynamics of CC chemokine receptor 7 (CCR7) bound to its native biased ligands, CCL19 and CCL21, and detect a series of molecular switches that are mediated by various ligand-induced allosteric events. These molecular switches involve three tyrosine residues (Y112(3.32), Y255(6.51), and Y288(7.39)), three phenylalanine residues (F116(3.36), F208(5.47), and F248(6.44)), and a polar interaction between Q252(6.48) and R294(7.45) in the transmembrane domain of CCR7. Conformational changes within these switches, particularly hydrogen bond formation between Y112(3.32) and Y255(6.51), lead to global helical movements in the receptor's transmembrane helices and contribute to the transitioning of the receptor to distinct states. Ligand-induced helical movements in the receptor highlight the ability of biased ligands to stabilize the receptor in different states through a dynamic network of allosteric events.

Hybrid flagellin as a T cell independent vaccine scaffold.

BACKGROUND: To extend the potency of vaccines against infectious diseases, vaccines should be able to exploit multiple arms of the immune system. One component of the immune system that is under-used in vaccine design is the subset of B cells known to be capable of responding to repetitive antigenic epitopes and differentiate into plasma cells even in the absence of T cell help (T-independent, TI). RESULTS: To target vaccine responses from T-independent B cells, we reengineered a bacterial Flagellin (FliC) by replacing its exposed D3 domain with a viral envelope protein from Dengue virus (DENV2). The resulting hybrid FliC protein (hFliC) was able to form stable filaments decorated with conformationally intact DENV2 envelope domains. These filaments were not only capable of inducing a T cell-dependent (TD) humoral antibody response, but also significant IgM and IgG3 antibody response in a helper T cell repertoire-restricted transgenic mouse model. CONCLUSIONS: Our results provide proof-of-principle demonstration that a reengineered hybrid FliC could be used as a platform for polymeric subunit vaccines, enhancing T cell-dependent and possibly inducing T-independent antibody responses from B-1 B cells as well.

In vivo directed differentiation of pluripotent stem cells for skeletal regeneration.

Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

Epithelial microvilli establish an electrostatic barrier to microbial adhesion.

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli ("microvillus-minus," or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions.

Deciphering the M-cell niche: insights from mouse models on how microfold cells "know" where they are needed.

Known for their distinct antigen-sampling abilities, microfold cells, or M cells, have been well characterized in the gut and other mucosa including the lungs and nasal-associated lymphoid tissue (NALT). More recently, however, they have been identified in tissues where they were not initially suspected to reside, which raises the following question: what external and internal factors dictate differentiation toward this specific role? In this discussion, we will focus on murine studies to determine how these cells are identified (e.g., markers and function) and ask the broader question of factors triggering M-cell localization and patterning. Then, through the consideration of unconventional M cells, which include villous M cells, Type II taste cells, and medullary thymic epithelial M cells (microfold mTECs), we will establish the M cell as not just a player in mucosal immunity but as a versatile niche cell that adapts to its home tissue. To this end, we will consider the lymphoid structure relationship and apical stimuli to better discuss how the differing cellular programming and the physical environment within each tissue yield these cells and their unique organization. Thus, by exploring this constellation of M cells, we hope to better understand the multifaceted nature of this cell in its different anatomical locales.

Mucosal vaccine design and delivery.

Mucosal surfaces are a major portal of entry for many human pathogens that are the cause of infectious diseases worldwide. Vaccines capable of eliciting mucosal immune responses can fortify defenses at mucosal front lines and protect against infection. However, most licensed vaccines are administered parenterally and fail to elicit protective mucosal immunity. Immunization by mucosal routes may be more effective at inducing protective immunity against mucosal pathogens at their sites of entry. Recent advances in our understanding of mucosal immunity and identification of correlates of protective immunity against specific mucosal pathogens have renewed interest in the development of mucosal vaccines. Efforts have focused on efficient delivery of vaccine antigens to mucosal sites that facilitate uptake by local antigen-presenting cells to generate protective mucosal immune responses. Discovery of safe and effective mucosal adjuvants are also being sought to enhance the magnitude and quality of the protective immune response.

Convergent and divergent development among M cell lineages in mouse mucosal epithelium.

M cells are specialized epithelial cells mediating immune surveillance of the mucosal lumen by transepithelial delivery of Ags to underlying dendritic cells (DC). At least three M cell phenotypes are known in the airways and intestine, but their developmental relationships are unclear. We used reporter transgenic mouse strains to follow the constitutive development of M cell subsets and their acute induction by cholera toxin (CT). M cells overlying intestinal Peyer's patches (PPs), isolated lymphoid follicles, and nasal-associated lymphoid tissue are induced by distinct settings, yet show convergent phenotypes, such as expression of a peptidoglycan recognition protein-S (PGRP-S) transgene reporter. By contrast, though PP, isolated lymphoid follicle, and villous M cells are all derived from intestinal crypt stem cells, their phenotypes were clearly distinct; for example, PP M cells frequently appeared to form M cell-DC functional units, whereas villous M cells did not consistently engage underlying DC. B lymphocytes are critical to M cell function by forming a basolateral pocket and possible signaling through CD137; however, initial commitment to all M cell lineages is B lymphocyte and CD137 independent. CT causes induction of new M cells in the airway and intestine without cell division, suggesting transdifferentiation from mature epithelial cells. In contrast with intestinal PP M cells, CT-induced nasal-associated lymphoid tissue M cells appear to be generated from ciliated Foxj1(+)PGRP-S(+) cells, indicative of a possible precommitted progenitor. In summary, constitutive and inducible differentiation of M cells is toward strictly defined context-dependent phenotypes, suggesting specialized roles in surveillance of mucosal Ags.

The seed and the soil: optimizing stem cells and their environment for tissue regeneration.

The potential for stem cells to serve as cellular building blocks for reconstruction of complex defects has prompted significant enthusiasm in the field of regenerative medicine. Clinical application, however, is still limited, as implantation of cells into hostile wound environments may greatly hinder their tissue forming capacity. To circumvent this obstacle, novel approaches have been developed to manipulate both the stem cell itself and its surrounding environmental niche. By understanding this paradigm of seed and soil optimization, innovative strategies may thus be developed to harness the true promise of stem cells for tissue regeneration.

Route of administration significantly affects particle deposition and cellular recruitment.

Lung exposures to dusts, pollutants, and other aerosol particulates are known to be associated with pulmonary diseases such as asthma and Chronic Obstructive Pulmonary Disease. These health impacts are attributed to the ability of aerosol components to induce pulmonary inflammation, which promotes tissue remodeling, including fibrosis, tissue degradation, and smooth muscle proliferation. Consequently, the distribution of these effects can have a significant impact on the physiologic function of the lung. In order to study the impact of distribution of inhaled particulates on lung pathogenesis, we compared the effect of different methods of particle delivery. By comparing intranasal versus aerosol delivery of fluorescent microspheres, we observed strikingly distinct patterns of particle deposition; intranasal delivery provided focused deposition concentrated on larger airways, while aerosol delivery showed unform deposition throughout the lung parenchyma. Recognizing that the impacts of inflammatory cells are contingent upon their recruitment and behavior, we postulate that these variations in distribution patterns can result in significant alterations in biological responses. To elucidate the relevance of these findings in terms of biological representation, we subsequently conducted an investigation into the responses elicited by the administration of endotoxin (bacterial Lipopolysaccharide, or LPS) in a transgenic neutrophil reporter mouse model. As with the microsphere results, patterns of recruited neutrophil inflammatory responses matched the delivery method; that is, despite the active migratory behavior of neutrophils, inflammatory histopathology patterns were either focused on large airways (intranasal administration) or diffusely throughout the parenchyma (aerosol). These results demonstrate the importance of modes of aerosol delivery as different patterns of inflammation and tissue remodeling will have distinct impacts on lung physiology.

Aerosolized aqueous dust extracts collected near a drying lake trigger acute neutrophilic pulmonary inflammation reminiscent of microbial innate immune ligands.

BACKGROUND: A high incidence of asthma is prevalent among residents near the Salton Sea, a large inland terminal lake in southern California. This arid region has high levels of ambient particulate matter (PM); yet while high PM levels are often associated with asthma in many environments, it is possible that the rapidly retreating lake, and exposed playa or lakebed, may contribute components with a specific role in promoting asthma symptoms. OBJECTIVES: Our hypothesis is that asthma may be higher in residents closest to the Salton Sea due to chronic exposures to playa dust. Playa emissions may be concentrating dissolved material from the lake, with microbial components capable of inducing pulmonary innate immune responses. To test this hypothesis, we used a mouse model of aerosol exposures to assess the effects of playa dust. METHODS: From dust collected around the Salton Sea region, aqueous extracts were used to generate aerosols, which were injected into an environmental chamber for mouse exposure studies. We compared the effects of exposure to Salton Sea aerosols, as well as to known immunostimulatory reference materials. Acute 48-h and chronic 7-day exposures were compared, with lungs analyzed for inflammatory cell recruitment and gene expression. RESULTS: Dust from sites nearest to the Salton Sea triggered lung neutrophil inflammation that was stronger at 48-h but reduced at 7-days. This acute inflammatory profile and kinetics resembled the response to innate immune ligands LTA and LPS while distinct from the classic allergic response to Alternaria. CONCLUSION: Lung inflammatory responses to Salton Sea dusts are similar to acute innate immune responses, raising the possibility that microbial components are entrained in the dust, promoting inflammation. This effect highlights the health risks at drying terminal lakes from inflammatory components in dust emissions from exposed lakebed.

Scarless fetal skin wound healing update.

Scar formation, a physiologic process in adult wound healing, can have devastating effects for patients; a multitude of pathologic outcomes, affecting all organ systems, stems from an amplification of this process. In contrast to adult wound repair, the early-gestation fetal skin wound heals without scar formation, a phenomenon that appears to be intrinsic to fetal skin. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults. Unique properties of fetal cells, extracellular matrix, cytokine profile, and gene expression contribute to this scarless repair. Despite the great increase in knowledge gained over the past decades, the precise mechanisms regulating scarless fetal healing remain unknown. Herein, we describe the current proposed mechanisms underlying fetal scarless wound healing in an effort to recapitulate the fetal phenotype in the postnatal environment.