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

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

A Role for Syntaxin 3 in the secretion of IL-6 from Dendritic Cells following activation of Toll-like Receptors

The role of dendritic cells (DCs) in directing the immune response is due in part to their capacity to produce a range of cytokines. Importantly, DCs are a source of cytokines which can promote T cell survival and T helper cell differentiation. While it has become evident that soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors (SNARE) are involved in membrane fusion and ultimately cytokine release, little is known about which members of this family facilitate the secretion of specific cytokines from DCs. We profiled mRNA of eighteen SNARE proteins in DCs in response to activation with a panel of three toll-receptor (TLR) ligands and show differential expression of SNAREs in response to their stimulus and subsequent secretion patterns. Of interest, STX3 mRNA was up-regulated in response to TLR4 and TLR7 activation but not TLR2 activation. This correlated with secretion of IL-6 and MIP-1α. Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels. Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators. Given the role of IL-6 in Th17 differentiation, these findings suggest the potential of STX3 as therapeutic target in inflammatory disease

The emerging role of SNARE proteins in dendritic cell function

Dendritic cells (DCs) provide an essential link between innate and adaptive immunity. At the site of infection, antigens recognised by DCs via pattern-recognition receptors such as Toll like receptors (TLR), initiate a specific immune response. Depending on the nature of the antigen, DCs secrete distinct cytokines with which they orchestrate homeostasis and pathogen clearance. Dysregulation of this process can lead to unnecessary inflammation which can result in a plethora of inflammatory diseases. Therefore, the secretion of cytokines from DCs is tightly regulated and this regulation is facilitated by highly conserved trafficking protein families. These proteins control the transport of vesicles from the golgi complex to the cell surface and between organelles. In this review we will discuss the role of soluble n-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs) in DCs both as facilitators of secretion and as useful tools to determine the pathways of secretion through their definite locations within the cells and inherent specificity in opposing binding partners on vesicles and target membranes. The role of SNAREs in DC function may present an opportunity to explore these proteins as novel targets in inflammatory disease