Immune Mediated Shaping of Microflora Community Composition Depends on Barrier Site

Barrier surfaces, such as the intestinal lining and the skin, are colonized by a diverse community of commensal microorganisms. Although commensal microorganisms clearly impact the host immune system, whether the immune system also shapes the commensal community is poorly understood. We used 16S rDNA deep sequencing to test whether mice with specific immune defects have an altered commensal microflora. Initially, skin swabs were obtained from wild-type and Langerhans Cell (LC) deficient mice. Despite the intimate contacts that LC make with the upper epidermis, no significant differences were observed in microbial community composition. Similarly, the skin of MyD88/TRIF^−/−, Rag1^−/− and heterozygous littermate controls showed no alteration in their commensal communities. Next we examined mouth swabs and feces. We did not find a difference in the MyD88/TRIF^−/− mice. However, we did observe a significant shift in the microbial composition in the feces and mouths of Rag1^−/− mice. Thus, we conclude that the adaptive immune system modulates the microbial composition at mucosal surfaces in the steady-state but LC, adaptive immunity, and MyD88-dependent innate responses do not affect the skin microbiome revealing a major distinction between barrier sites.     

Commensal pathogens, with a focus on Streptococcus pneumoniae, and interactions with the human host

Many important pathogens have humans as their normal ecological niche where healthy carriage dominates over disease. The ability of these commensal pathogens, such as Streptococcus pneumoniae, to cause disease depends on a series of microbial factors as well as of genetic and environmental factors in the human host affecting the clearing capacity mediated by the innate and adaptive immune system. This delicate interplay between microbe and host affects not only the likelihood for a commensal pathogen to cause disease, but also disease type and disease severity.     

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer''s patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c⁺CD11b⁺CD8⁻ dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.     

Differential induction of innate memory in porcine monocytes by β-glucan or bacillus Calmette-Guerin

Innate immunomodulation via induction of innate memory is one mechanism to alter the host’s innate immune response to reduce or prevent disease. Microbial products modulate innate responses with immediate and lasting effects. Innate memory is characterized by enhanced (training) or depressed (tolerance) innate immune responses, including pro-inflammatory cytokine production, to secondary exposure following a priming event. To investigate the ability of β-glucans and bacillus Calmette-Guerin to induce innate training or tolerance in pig cells, porcine monocytes were cultured with priming agonist (β-glucans or bacillus Calmette-Guerin) then re-stimulated 5 d later with a heterologous microbial agonist to determine induction of innate memory. Priming with β-glucan from Saccharomyces cerevisiae depressed IL-1β and TNF-α cytokine responses to re-stimulation with LPS, indicative of a tolerized state. However, bacillus Calmette-Guerin priming induced a trained state in porcine monocytes, as LPS re-stimulation enhanced IL-1β and TNF-α gene expression and protein production. We present the first evidence of innate memory in pig monocytes, with bacillus Calmette-Guerin (training) or Saccharomyces cerevisiae β-glucan (tolerance). Induction of a trained or tolerized state in vitro is a first step to identify agonists to alter the innate immune system at the animal level with the intent of enhancing disease resistance.     

Saccharomyces cerevisiae as a probiotic feed additive to non and pseudo-ruminant feeding: a review

The production of livestock and poultry faces major challenges to meet the global demand for meat and dairy products and eggs due to a steady increase in the world’s population and the ban of antibiotics in animal production. This ban has forced animal nutritionists to seek for natural alternatives to antibiotics. In this context, the yeast Saccharomyces cerevisiae has received considerable attention in the last decade. It has been reported that feed supplementation with live yeast cells improve feed efficiency, enhance feed digestibility, increase animal performance, reduce the number of pathogenic bacteria, improve animal health and reduce the negative environmental impacts of livestock production. The current review sheds light on the effects of the use of live S. cerevisiae cells in the diets of nonruminant and pseudo-ruminant’s animals and the mechanisms by which they exert its effects. This review work revealed that the addition of S. cerevisiae in poultry feed causes a phenomenon called competitive exclusion of pathogenic bacteria capable of causing disease adhere to the yeast surface, and so removing a large amount of harmful micro-organisms and allowing the Animal defend more effectively, the production of antimicrobial agents, the balancing the gut microbiota and stimulation of host adaptive immune system and improving gut morphological structure, thus these benefits are reflected on the overall poultry health. In addition, in the presence of live S. cerevisiae cells, the immunity of rabbits was improved due to the high number of white blood cell. In addition, apparent digestibility of acid and neutral detergent fibre was improved in horses and rabbits. Saccharomyces cerevisiae in pig diets augment mucosal immunity by increasing IgM and IgA activity against pathogens, enhance intestinal development and function, adsorb mycotoxins, modulate gut microbiota and reduce postweaning diarrhoea.     

Sex differences in the protection of host immune systems by a polyembryonic parasitoid

Endoparasitoids have the ability to evade the cellular immune responses of a host and to create an environment suitable for survival of their progeny within a host. Generally, the host immune system is suppressed by endoparasitoids. However, polyembryonic endoparasitoids appear to invade their hosts using molecular mimicry rather than immune system suppression. It is not known how the host immune system is modified by polyembryonic endoparasitoids. Using haemocyte counts and measurement of cellular immune responses, we evaluated modification of the host immune system after separate infestations by a polyembryonic parasitoid (Copidosoma floridanum) and another parasitoid (Glyptapanteles pallipes) and by both together (multi-parasitism). We found that the polyembryonic parasitoid maintains and enhances the host immune system, whereas the other parasitoid strongly suppresses the immune system. Multi-parasitization analysis revealed that C. floridanum cancelled the immune suppression by G. pallipes and strengthened the host immunity. This enhancement was much stronger with male than with female C. floridanum.     

Predation on Multiple Trophic Levels Shapes the Evolution of Pathogen Virulence

The pathogen virulence is traditionally thought to co-evolve as a result of reciprocal selection with its host organism. In natural communities, pathogens and hosts are typically embedded within a web of interactions with other species, which could affect indirectly the pathogen virulence and host immunity through trade-offs. Here we show that selection by predation can affect both pathogen virulence and host immune defence. Exposing opportunistic bacterial pathogen Serratia marcescens to predation by protozoan Tetrahymena thermophila decreased its virulence when measured as host moth Parasemia plantaginis survival. This was probably because the bacterial anti-predatory traits were traded off with bacterial virulence factors, such as motility or resource use efficiency. However, the host survival depended also on its allocation to warning signal that is used against avian predation. When infected with most virulent ancestral bacterial strain, host larvae with a small warning signal survived better than those with an effective large signal. This suggests that larval immune defence could be traded off with effective defence against bird predators. However, the signal size had no effect on larval survival when less virulent control or evolved strains were used for infection suggesting that anti-predatory defence against avian predators, might be less constrained when the invading pathogen is rather low in virulence. Our results demonstrate that predation can be important indirect driver of the evolution of both pathogen virulence and host immunity in communities with multiple species interactions. Thus, the pathogen virulence should be viewed as a result of both past evolutionary history, and current ecological interactions.     

Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells

Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.     

Pichia acaciae Killer System: Genetic Analysis of Toxin Immunity

The gene responsible for self-protection in the Pichia acaciae killer plasmid system was identified by heterologous expression in Saccharomyces cerevisiae. Resistance profiling and conditional toxin/immunity coexpression analysis revealed dose-independent protection by pPac1-2 ORF4 and intracellular interference with toxin function, suggesting toxin reinternalization in immune killer cells.     

Phage gene expression and host responses lead to infection-dependent costs of CRISPR immunity

CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.Subject terms: Bacteriophages, Microbial ecology     

Innate immune recognition of microbial cell wall components and microbial strategies to evade such recognitions

The innate immune system constitutes the first line of defence against invading microbes. The basis of this defence resides in the recognition of defined structural motifs of the microbes called “Microbial associated molecular patterns” that are absent in the host. Cell wall, the outer layer of both bacterial and fungal cells, a unique structure that is absent in the host and is recognized by the germ line encoded host receptors. Nucleotide oligomerization domain proteins, peptidoglycan recognition proteins and C-type lectins are host receptors that are involved in the recognition of bacterial cell wall (usually called peptidoglycan), whereas fungal cell wall components (N- and O-linked mannans, β-glucans etc.) are recognized by host receptors like C-type lectins (Dectin-1, Dectin-2, mannose receptor, DC-SIGN), Toll like receptors-2 and -4 (TLR-2 and TLR-4). These recognitions lead to activation of a variety of host signaling cascades and ultimate production of anti-microbial compounds including phospholipase A2, antimicrobial peptides, lysozyme, reactive oxygen and nitrogen species. These molecules act in cohort against the invading microbes to eradicate infections. Additionally pathogen recognition leads to the production of cytokines, which further activate the adaptive immune system. Both pathogenic and commensal bacteria and fungus use numerous strategies to subvert the host defence. These strategies include bacterial peptidoglycan glycan backbone modifications by O-acetylation, N-deacetylation, N-glycolylation and stem peptide modifications by amidation of meso-Diaminopimelic acid; fungal cell wall modifications by shielding the β-glucan layer with mannoproteins and α-1,3 glucan. This review focuses on the recent advances in understanding the role of bacterial and fungal cell wall in their innate immune recognition and evasion strategies.     

Food-mediated modulation of immunity in a phytophagous insect: An effect of nutrition rather than parasitic contamination

Inherent to the cost of immunity, the immune system itself can exhibit tradeoffs between its arms. Phytophagous insects face a wide range of microbial and eukaryotic parasites, each activating different immune pathways that could compromise the activity of the others. Feeding larvae are primarily exposed to microbes, which growth is controlled by antibiotic secondary metabolites produced by the host plant. The resulting variation in abundance of microbes on plants is expected to differentially stimulate the insect antimicrobial immune defenses. Under the above tradeoff hypothesis, stimulation of the insect antimicrobial defenses is expected to compromise immune activity against eukaryote parasites. In the European grape berry moth, Eupoecilia ambiguella, immune effectors directed towards microbes are negatively correlated to those directed towards eukaryotic parasites among host plants. Here, we hypothesize this relationship is caused by a variable control of the microbial community among host plants by their antibiotic metabolites. To test this hypothesis, we first quantified antimicrobial activity in berries of several grape varieties. We then measured immune defenses of E. ambiguella larvae raised on artificial diets in which we mimicked levels of antimicrobial activity of grape berries using tetracycline to control the abundance of growing microbes. Another group of larvae was raised on artificial diets made of berry extracts only to control for the effect of nutrition. We found that controlling microbe abundance with tetracycline in diets did not explain variation in the immune function whereas the presence of berry extracts did. This suggests that variation in immune defenses of E. ambiguella among grape varieties is caused by nutritional difference among host plants rather than microbe abundance. Further study of the effects of berry compounds on larval immune parameters will be needed to explain the observed tradeoff among immune system components.     

Regulatory T Cells and Immune Tolerance in the Intestine

A fundamental role of the mammalian immune system is to eradicate pathogens while minimizing immunopathology. Instigating and maintaining immunological tolerance within the intestine represents a unique challenge to the mucosal immune system. Regulatory T cells are critical for continued immune tolerance in the intestine through active control of innate and adaptive immune responses. Dynamic adaptation of regulatory T-cell populations to the intestinal tissue microenvironment is key in this process. Here, we discuss specialization of regulatory T-cell responses in the intestine, and how a breakdown in these processes can lead to chronic intestinal inflammation.The mammalian host harbors a vast and diverse commensal microbiota. The gastrointestinal tract is a site of preferential colonization by commensal organisms, consisting of fungal, viral, and bacterial species. Initial microbial colonization of the host occurs during birth and continues until a stable commensal microbiota is established during childhood (Tannock 2007). Colonization of the gastrointestinal tract is a vital triggering stimuli for maturation of the mucosal immune system, and the presence of a commensal microbiota further benefits the host by providing resistance to invading pathogens and metabolism of dietary components (Macpherson et al. 2005; Hooper et al. 2012). A dynamic molecular dialogue between microbiota and host ensures this colonization occurs as a state of mutualism, the breakdown of which can result in chronic pathologies of the gastrointestinal tract, such as inflammatory bowel diseases (IBD) (Kaser et al. 2010; Maloy and Powrie 2011). Complex interactions between the microbiota, mucosal immune system, and the intestinal tissue cells provide multiple layers of regulation that control intestinal immunity. Here, we focus on the role of regulatory T cells as key components of intestinal homeostasis and discuss how tissue-specific adaptations contribute to their function when patrolling this challenging frontier.     

Vibrio cholerae Proteome-Wide Screen for Immunostimulatory Proteins Identifies Phosphatidylserine Decarboxylase as a Novel Toll-Like Receptor 4 Agonist

Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called “EPSIA”, Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFα and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced ∼40% and ∼15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.