The emerging roles of intestinal macrophages in sickness and in health

The immune system in the intestine represents a unique environment that can quickly respond to harmful pathogens, but it remains tolerant to antigens from food and commensal bacteria. This balance between protective immunity and tolerance is largely dependent on the mononuclear phagocytes in the intestine, such as macrophages. Intestinal macrophages, unlike other macrophage populations in the body, are hypo-responsive to stimuli although they originate from fully responsive blood monocytes. The intestinal environment seems to instruct monocytes to mute their function upon arrival to the gut in order to adapt to the antigen-rich environment. While their main role in the healthy gut is to maintain homeostasis, in disease macrophage phenotype and function is changed and these cells become the drivers of inflammation and disease progression

Modulation of the Nuclear Transcription Factor of Activated T Cells by Duck Hepatitis B Virus

During infection with hepadnaviruses besides the infectious agent a high number of subviral particles without nucleocapsids are produced, which are able to change the infection dramatically. In addition, it was observed that the activation of the nuclear factor of activated T cells, regulated usually in cells of the immune system, was strongly influenced after infection. When primary duck liver cells were infected with purified virions of duck hepatitis B virus the activation of this factor was reduced in a similar way as it was achieved by inhibition of calcineurin, a cellular phosphatase necessary to control the factor, whereas the addition of subviral particles inhibited this reduction. It was found that the large surface protein of the virus was responsible for the reduced activity. Although this protein was embedded in similar amounts into the envelopes of both particles, only virions were able to inhibit the activity of the nuclear factor. An explanation of the different performances of the particles in primary duck liver cells apparently depends on the individual mode of insertion of the large surface proteins into the viral membrane. Furthermore, the nuclear factor of activated T cells could only be detected in liver sinusoidal endothelial cells, which was shown being attracted by virions but not by subviral particles

Patulin, deoxynivalenol, zearalenone and T-2 toxin affect viability and modulate cytokine secretion in J774A.1 murine macrophages

Mycotoxins are secondary fungal metabolites, which occur in food and feed. They have detrimental effects on the health of humans and animals, and they are known to cause immunosuppression. In this study the effect of patulin, deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin exposure on the viability and the secretion of key pro- and anti-inflammatory cytokines from the murine macrophage cell line, J774A.1, was investigated. Exposure of macrophages to high doses of ZEN (100,000 pg/mL) and T-2 toxin (10,000 and 100,000 pg/mL) resulted in a significant decrease (P < 0.05 and P < 0.01) in cell viability. Exposure of macrophages to these mycotoxins resulted in a dose-dependent modulation of cytokine secretion. Specifically, exposure to low doses of patulin (0.001, 0.1 and 1 pg/mL) resulted in a statistically significant decrease in the secretion of the pro-inflammatory cytokines interleukin (IL) 6 (IL-6) and tumor necrosis factor alpha (TNF-α), following stimulation with lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls. Treatment with low doses of DON (0.001 pg/mL) and ZEN (0.001 and 0.01 pg/mL) significantly decreased (P < 0.01) the secretion of the pro-inflammatory cytokine IL-12p40, while several doses of T-2 toxin (0.001, 0.01, 0.1, 1 and 100 pg/mL) caused a significant decrease the expression of IL-6. Each of the mycotoxins also significantly increased the production of the anti-inflammatory cytokine IL-10, both before and after LPS stimulation. This data provides further insight into the mechanisms by which mycotoxins modulate the host immune response to exert their immunosuppressive activity

Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection

Profiling humoral immune responses to Clostridium difficile-specific antigens by protein microarray analysis

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated<10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost

Protective antibodies against Clostridium difficile are present in intravenous immunoglobulin and are retained in humans following its administration

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation

A Role for TLR4 in Clostridium difficile Infection and the Recognition of Surface Layer Proteins

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system

Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences

Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53R273H in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53R273H targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin