Differential cytokine response from dendritic cells to commensal and pathogenic bacteria in different lymphoid compartments in humans

Resident host microflora condition and prime the immune system. However, systemic and mucosal immune responses to bacteria may be divergent. Our aim was to compare, in vitro, cytokine production by human mononuclear and dendritic cells (DCs) from mesenteric lymph nodes (MLNs) and peripheral blood mononuclear cells (PBMCs) to defined microbial stimuli. Mononuclear cells and DCs isolated from the MLN (n = 10) and peripheral blood (n = 12) of patients with active colitis were incubated in vitro with the probiotic bacteria Lactobacillus salivarius UCC118 or Bifidobacterium infantis 35624 or the pathogenic organism Salmonella typhimurium UK1. Interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and IL-10 cytokine levels were quantified by ELISA. PBMCs and PBMC-derived DCs secreted TNF-alpha in response to the Lactobacillus, Bifidobacteria, and Salmonella strains, whereas MLN cells and MLN-derived DCs secreted TNF-alpha only in response to Salmonella challenge. Cells from the systemic compartment secreted IL-12 after coincubation with Salmonella or Lactobacilli, whereas MLN-derived cells produced IL-12 only in response to Salmonella. PBMCs secreted IL-10 in response to the Bifidobacterium strain but not in response to the Lactobacillus or Salmonella strain. However, MLN cells secreted IL-10 in response to Bifidobacteria and Lactobacilli but not in response to Salmonella. In conclusion, commensal bacteria induced regulatory cytokine production by MLN cells, whereas pathogenic bacteria induce T cell helper 1-polarizing cytokines. Commensal-pathogen divergence in cytokine responses is more marked in cells isolated from the mucosal immune system compared with PBMCs.     

Systemic control of plasmacytoid dendritic cells by CD8+ T cells and commensal microbiota

The composition of the intestinal microbial community is a distinctive individual trait that may divergently influence host biology. Because dendritic cells (DC) regulate the quality of the host response to microbiota, we evaluated DC in mice bearing distinct enteric microbial communities divergent for colitis susceptibility. Surprisingly, a selective, systemic reduction of plasmacytoid dendritic cells (pDC) was observed in isogenic mice with different microbiota: restricted flora (RF) vs specific pathogen free (SPF). This reduction was not observed in germfree mice, suggesting that the pDC deficiency was not simply due to a lack of intestinal microbial products. The microbial action was linked to cytotoxic CD8(+) T cells, since pDC in RF mice were preserved in the CD8(-/-) and perforin(-/-) genotypes, partially restored by anti-CD8beta Ab, and augmented in SPF mice bearing the TAP(-/-) genotype. Direct evidence for pDC cytolysis was obtained by rapid and selective pDC depletion in SPF mice transferred with RF CD8(+) T cells. These data indicate that commensal microbiota, via CTL activation, functionally shape systemic immune regulation that may modify risk of inflammatory disease.     

Role of the commensal microbiota in normal and pathogenic host immune responses

The commensal microbiota that inhabit different parts of the gastrointestinal (GI) tract have been shaped by coevolution with the host species. The symbiotic relationship of the hundreds of microbial species with the host requires a tuned response that prevents host damage, e.g., inflammation, while tolerating the presence of the potentially beneficial microbes. Recent studies have begun to shed light on immunological processes that participate in maintenance of homeostasis with the microbiota and on how disturbance of host immunity or the microbial ecosystem can result in disease-provoking dysbiosis. Our growing appreciation of this delicate host-microbe relationship promises to influence our understanding of inflammatory diseases and infection by microbial pathogens and to provide new therapeutic opportunities.     

Altered microbiota associated with abnormal humoral immune responses to commensal organisms in enthesitis-related arthritis

Introduction

Prior studies have established altered microbiota and immunologic reactivity to enteric commensal organisms in inflammatory bowel disease (IBD). Since intestinal inflammation is present in a subset of patients with both pediatric and adult spondyloarthritis (SpA), we hypothesized that SpA patients may also have altered microbiota and immune responsiveness to enteric organisms.

Methods

Stool and blood specimens were collected from children with enthesitis-related arthritis (ERA) and non-inflammatory controls. DNA purified from stool was subject to PCR amplification and sequencing of the variable IV region from the 16S rDNA gene. IgA and IgG Enzyme-linked Immunosorbent Assays (ELISAs) were performed on select species of bacteria in most subjects.

Results

Twenty-five children with ERA and 13 controls were included. The ERA patients had less Faecalibacterium prausnitzii (3.8% versus 10%, P = 0.008) and lachnospiraceae family (12 versus 7.0%, P = 0.020), a statistically significant increase in bifidobacterium (1.8% versus 0%, P = 0.032) and a non-statistically significant increase in Bacteroides (21% versus 11%, P = 0.150). Akkermansia muciniphila was abundant (>2%) in 7/27 ERA patients but none of the controls (P = 0.072.) Cluster analysis revealed two clusters of ERA patients: Cluster one (n = 8) was characterized by high levels of Bacteroides genus, while a second (n = 15) cluster had similar levels as the controls. Seven of 17 (41%) of the ERA subjects in Cluster 2 compared to 0/8 of the subjects in Cluster 1 had abundant Akkermansia muciniphila (P = 0.057). Serum IgA and IgG antibody levels against F. prausnitzii and B. fragilis were similar between patients and controls, whereas the two groups showed divergent responses when the fecal relative abundances of F. prausnitzii and Bacteroides were compared individually against IgA antibody levels recognizing F. prausnitzii and B. fragilis, respectively.

Conclusion

The abundance of F. prausnitzii in the stool among patients with ERA is reduced compared to controls, and Bacteroides and A. muciniphila are identified as associative agents in subsets of ERA patients. Differences in the humoral responses to these bacteria may contribute to disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0486-0) contains supplementary material, which is available to authorized users.     

Divergent response to LPS and bacteria in CD14-deficient murine macrophages

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.     

Potentially pathogenic bacteria cultured from the sputum of stable asthmatics are associated with increased 8-isoprostane and airway neutrophilia

Abstract

Potential bacterial pathogens are found in the airways in several diseases that are associated with neutrophilic inflammation. The aim of this study was to characterize subjects with stable asthma, with no symptoms of respiratory infection, to assess whether key potentially pathogenic bacteria were present in significant quantities in the airways and to correlate this with the pattern of airway inflammation and oxidative stress. Subjects with stable asthma (n = 115) and healthy controls (n = 8) underwent clinical assessment, including hypertonic saline challenge combined with sputum induction. A significant load of potentially pathogenic bacteria (> 10⁶ cfu/mL) was cultured from the sputum of 17 (15%) subjects with stable asthma and was associated with higher total cell counts, proportion and number of neutrophils, sputum IL-8 and 8-isoprostane concentrations. The role of bacteria in potentiating neutrophilic asthma warrants further investigation. Therapies such as antibiotic and antioxidant treatment may be most effective in this sub-group of patients.     

Erythrophore cell response to food‐associated pathogenic bacteria: implications for detection

Cell‐based biosensors have been proposed for use as function‐based detectors of toxic agents. We report the use of Betta splendens chromatophore cells, specifically erythrophore cells, for detection of food‐associated pathogenic bacteria. Evaluation of erythrophore cell response, using Bacillus spp., has revealed that this response can distinguish pathogenic Bacillus cereus from a non‐pathogenic B. cereus ΔplcR deletion mutant and a non‐pathogenic Bacillus subtilis. Erythrophore cells were exposed to Salmonella enteritidis, Clostridium perfringens and Clostridium botulinum. Each bacterial pathogen elicited a response from erythrophore cells that was distinguished from the corresponding bacterial growth medium, and this observed response was unique for each bacterial pathogen. These findings suggest that erythrophore cell response has potential for use as a biosensor in the detection and toxicity assessment for food‐associated pathogenic bacteria.     

羧基荧光素脂质体的制备及其与病原菌的响应行为

【背景】由于目前临床上检测细菌感染的方法既耗时又昂贵,所以开发快速简便的鉴别方法势在必行。【目的】通过在脂质体膜材料中添加不同的稳定剂,研究它们对于脂质体稳定性的影响,并探究荧光素脂质体与病原菌之间的响应情况。【方法】以磷脂酰胆碱和胆固醇为主要原料,1,2-棕榈酰磷脂酰甘油[1,2-Dipalmitoyl-sn-glycero-3-phospho-(1?-rac-glycerol),DPPG]、十八胺和10,12-二十三二炔酸(10,12-Tricosadiyonic acid,TCDA)为稳定剂,采用薄膜分散-超声法制备羧基荧光素脂质体。利用病原菌能够分泌毒力因子造成脂质体渗漏的原理,将病原菌菌液和上清液分别与荧光素脂质体孵育,检测从脂质体渗漏的羧基荧光素的荧光强度,反映病原菌的毒性程度。【结果】DPPG和TCDA都能增加脂质体的稳定性,而十八胺的添加则导致脂质体稳定性的降低。与金黄色葡萄球菌ATCC25923和铜绿假单胞菌PAO1菌液和上清液共同孵育的脂质体荧光强度大量增加,与大肠杆菌DH5α和PBS缓冲液共同孵育的脂质体荧光强度几乎不变。【结论】能够分泌外毒素的金黄色葡萄球菌和铜绿假单胞菌都能对脂质体产生响应,而不分泌外毒素的大肠杆菌则不会对脂质体产生响应。     

Establishment of tolerance to commensal bacteria requires a complex microbiota and is accompanied by decreased intestinal chemokine expression

Intricate regulation of tolerance to the intestinal commensal microbiota acquired at birth is critical. We hypothesized that epithelial cell tolerance toward early gram-positive and gram-negative colonizing bacteria is established immediately after birth, as has previously been shown for endotoxin. Gene expression in the intestine of mouse pups born to dams that were either colonized with a conventional microbiota or monocolonized (Lactobacillus acidophilus or Eschericia coli) or germ free was examined on day 1 and day 6 after birth. Intestinal epithelial cells from all groups of pups were stimulated ex vivo with L. acidophilus and E. coli to assess tolerance establishment. Intestine from pups exposed to a conventional microbiota displayed lower expression of Ccl2, Ccl3, Cxcl1, Cxcl2, and Tslp than germ-free mice, whereas genes encoding proteins in Toll-like receptor signaling pathways and cytokines were upregulated. When comparing pups on day 1 and day 6 after birth, a specific change in gene expression pattern was evident in all groups of mice. Tolerance to ex vivo stimulation with E. coli was only established in conventional animals. Colonization of the intestine was reflected in the spleen displaying downregulation of Cxcl2 compared with germ-free animals on day 1 after birth. Colonization reduced the expression of genes involved in antigen presentation in the intestine-draining mesenteric lymph nodes, but not in the popliteal lymph nodes, as evidenced by gene expression on day 23 after birth. We propose that microbial detection systems in the intestine are upregulated by colonization with a diverse microbiota, whereas expression of proinflammatory chemokines is reduced to avoid excess recruitment of immune cells to the maturing intestine.     

Gut commensal bacteria,Paneth cells and their relations to radiation enteropathy

In steady state, the intestinal epithelium forms an important part of the gut barrier to defend against luminal bacterial attack. However, the intestinal epithelium is compromised by ionizing irradiation due to its inherent self-renewing capacity. In this process, small intestinal bacterial overgrowth is a critical event that reciprocally alters the immune milieu. In other words, intestinal bacterial dysbiosis induces inflammation in response to intestinal injuries, thus influencing the repair process of irradiated lesions. In fact, it is accepted that commensal bacteria can generally enhance the host radiation sensitivity. To address the determination of radiation sensitivity, we hypothesize that Paneth cells press a critical “button” because these cells are central to intestinal health and disease by using their peptides, which are responsible for controlling stem cell development in the small intestine and luminal bacterial diversity. Herein, the most important question is whether Paneth cells alter their secretion profiles in the situation of ionizing irradiation. On this basis, the tolerance of Paneth cells to ionizing radiation and related mechanisms by which radiation affects Paneth cell survival and death will be discussed in this review. We hope that the relevant results will be helpful in developing new approaches against radiation enteropathy.     

Consumption of Camembert cheese stimulates commensal enterococci in healthy human intestinal microbiota

Enterococci are natural inhabitants of the human gastrointestinal tract and the main Gram-positive and facultative anaerobic cocci recovered in human faeces. They are also present in a variety of fermented dairy and meat products, and some rare isolates are responsible for severe infections such as endocarditis and meningitis. The aim of the present study was to evaluate the effect of Camembert cheese consumption by healthy human volunteers on the faecal enterococcal population. A highly specific real-time quantitative PCR approach was designed and used to type enterococcal species in human faeces. Two species were found, Enterococcus faecalis and Enterococcus faecium, and only the Enterococcus faecalis population was significantly enhanced after Camembert cheese consumption, whereas Escherichia coli population and the dominant microbiota remained unaffected throughout the trial.     

Distinct commensal bacteria associated with ingesta and mucosal epithelium in the gastrointestinal tracts of calves and chickens

The primary aim of this study was to determine whether distinct gastrointestinal tract (GIT) microbial communities are established within ingesta and on mucosal surfaces of dairy calves and chickens to evaluate whether the principle of microbial segregation is of broad biological significance. Multivariate analysis of the predominant bacterial PCR-denaturing gradient gel electrophoresis profiles and estimated bacterial populations were compared in rumen, jejunum, ileum, cecum, and colon ingesta and matching mucosal tissues. Samples collected from 3-week old (n = 8) and 6-month old (n = 8) calves revealed that the predominant mucosa-associated bacteria were distinct from those inhabiting ingesta, and bacterial diversity varied significantly among the GIT regions. The estimated bacterial populations displayed significant regional differences for bovine mucosal (P = 0.05) and for ingesta (P = 0.03) only at 6 months of age. This indicates an established segregation of the enteric bacterial population throughout the GIT in weaned calves. Analysis of ileal and cecal bacterial profiles in chickens confirmed that the segregation of commensal bacteria between ingesta and the mucosal tissue was a common biological phenomenon. Our study provides some fundamental understanding of the impact of sample type (mucosa vs. ingesta), region, and host age on commensal bacterial establishment and segregation throughout the GIT.     

Oncostatin M production by human dendritic cells in response to bacterial products

Oncostatin M (OSM) is a pleiomorphic cytokine that belongs to the IL-6 cytokine family. It is produced by activated T cells and monocytes/macrophages and plays an important role in the process of inflammatory responses. Although dendritic cells (DCs) have been shown to secrete a variety of cytokines, it is not elucidated whether DCs are able to produce OSM. To clarify this, using human DCs derived from peripheral blood cells, we measured the protein levels of OSM in the supernatants of DC cultures by ELISA and examined the expression of OSM mRNA by RT-PCR after stimulation with lipopolysaccharide (LPS) or fixed Staphylococcus aureus (SACS). Upon stimulation with bacterial products, DCs secreted a large amount of OSM protein in a dose- and time-dependent manner. Concomitantly, the expression of OSM mRNA by DCs was markedly up-regulated. Compared the ability of DCs to produce OSM with that of monocytes, which are major producers of OSM, DCs released significantly higher amounts of OSM protein in the culture supernatants than monocytes. These findings indicate for the first time that human monocyte-derived DCs can synthesize and secrete large amounts of OSM in response to bacterial products, suggesting that OSM produced by DCs at infectious sites may play a role in modulating inflammatory responses.     

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.