Innate and adaptive autoimmunity in type 1 diabetes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74867/1/j.1399-5448.2007.00334.x.pd

The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis

Background: NOD2, an intracellular pathogen recognition sensor, modulates innate defences to muropeptides derived from various bacterial species, including Mycobacterium tuberculosis (MTB). Experimentally, NOD2 attenuates two key putative mycobactericidal mechanisms. TNF-alpha synthesis is markedly reduced in MTB-antigen stimulated-mononuclear cells expressing mutant NOD2 proteins. NOD2 agonists also induce resistance to apoptosis, and may thus facilitate the survival of MTB in infected macrophages. To further define a role for NOD2 in disease pathogenesis, we analysed NOD2 transcriptional responses in pulmonary leucocytes and mononuclear cells harvested from patients with pulmonary tuberculosis (PTB).Methods: We analysed NOD2 mRNA expression by real-time polymerase chain-reaction in alveolar lavage cells obtained from 15 patients with pulmonary tuberculosis and their matched controls. We compared NOD2 transcriptional responses, in peripheral leucocytes, before and after anti-tuberculous treatment in 10 patients. In vitro, we measured NOD2 mRNA levels in MTB-antigen stimulated-mononuclear cells.Results: No significant differences in NOD2 transcriptional responses were detected in patients and controls. In some patients, however, NOD2 expression was markedly increased and correlated with toll-like-receptor 2 and 4 expression. In whole blood, NOD2 mRNA levels increased significantly after completion of anti-tuberculosis treatment. NOD2 expression levels did not change significantly in mononuclear cells stimulated with mycobacterial antigens in vitro.Conclusion: There are no characteristic NOD2 transcriptional responses in PTB. Nonetheless, the increased levels of NOD2 expression in some patients with severe tuberculosis, and the increases in expression levels within peripheral leucocytes following treatment merit further studies in selected patient and control populations

Toll-like receptors in cellular subsets of human tonsil T cells: altered expression during recurrent tonsillitis

BACKGROUND: The palatine tonsils have a pivotal role in immunological detection of airborne and ingested antigens like bacteria and viruses. They have recently been demonstrated to express Toll-like receptors (TLRs), known to recognize molecular structures on such microbes and activate innate immune responses. Their activation might also provide a link between innate and adaptive immunity. In the present study, the expression profile of TLR1-TLR10 was characterized in human tonsil T cells, focusing on differences between subsets of CD4(+ )T helper (Th) cells and CD8(+ )cytotoxic T lymphocytes (CTL). The study was also designed to compare the TLR expression in T cells from patients with recurrent tonsillitis and tonsillar hyperplasia. METHODS: Tonsils were obtained from children undergoing tonsillectomy, and classified according to the clinical diagnoses and the outcome of tonsillar core culture tests. Two groups were defined; recurrently infected tonsils and hyperplastic tonsils that served as controls. Subsets of T cells were isolated using magnetic beads. The expression of TLR transcripts in purified cells was assessed using quantitative real-time RT-PCR. The corresponding protein expression was investigated using flow cytometry and immunohistochemistry. RESULTS: T cells expressed a broad repertoire of TLRs, in which TLR1, TLR2, TLR5, TLR9 and TLR10 predominated. Also, a differential expression of TLRs in CD4(+ )and CD8(+ )T cells was obtained. TLR1 and TLR9 mRNA was expressed to a greater extent in CD4(+ )cells, whereas expression of TLR3 mRNA and protein and TLR4 protein was higher in CD8(+ )cells. CD8(+ )cells from infected tonsils expressed higher levels of TLR2, TLR3 and TLR5 compared to control. In contrast, CD4(+ )cells exhibited a down-regulated TLR9 as a consequence of infection. CONCLUSION: The present study demonstrates the presence of a broad repertoire of TLRs in T cells, a differential expression in CD4(+ )and CD8(+ )cells, along with infection-dependent alterations in TLR expression. Collectively, these results support the idea that TLRs are of importance to adaptive immune cells. It might be that TLRs have a direct role in adaptive immune reactions against infections. Thus, further functional studies of the relevance of TLR stimulation on T cells will be of importance

Characterization analysis and polymorphism detection of the porcine Myd88 gene

The myeloid differentiation primary response protein 88 (Myd88) is an essential adaptor protein, which mediates in all Toll-like receptor (TLR) members signal transduction, except for TLR3. In this study, the 4464 bp genomic sequence of porcine Myd88 was first isolated, whereupon tissue distribution, chromosome mapping and single nucleotide polymorphism (SNP) were analyzed. Our results revealed that porcine Myd88 gene, which was located at chromosome 13 linked with marker S0288 (distance = 40 cR; LOD = 8.66), was widely expressed in all the examined tissues. There were 16 potential SNPs in the isolated genome fragment. SNP 797T/C in the first intron was studied, with no significant association being found between the genotype and immune traits in pigs (p > 0.05). The porcine Myd88 protein contained both the death domain (DD) and the Toll/IL-1 receptor domain (TIR). Leu residues, essential for its structure, were the most abundant encountered in the DD. The TIR contained two conserved motifs which may play important roles in the Myd88 function

Genes Differentially Expressed in Conidia and Hyphae of Aspergillus fumigatus upon Exposure to Human Neutrophils

Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans

In vivo expression of innate immunity markers in patients with mycobacterium tuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs), Coronin-1 and Sp110 are essential factors for the containment of <it>Mycobacterium tuberculosis </it>infection. The purpose of this study was to investigate the <it>in vivo </it>expression of these molecules at different stages of the infection and uncover possible relationships between these markers and the state of the disease.</p> <p>Methods</p> <p>Twenty-two patients with active tuberculosis, 15 close contacts of subjects with latent disease, 17 close contacts of subjects negative for mycobacterium antigens and 10 healthy, unrelated to patients, subjects were studied. Quantitative mRNA expression of Coronin-1, Sp110, TLRs-1,-2,-4 and -6 was analysed in total blood cells <it>vs </it>an endogenous house-keeping gene.</p> <p>Results</p> <p>The mRNA expression of Coronin-1, Sp110 and TLR-2 was significantly higher in patients with active tuberculosis and subjects with latent disease compared to the uninfected ones. Positive linear correlation for the expression of those factors was only found in the infected populations.</p> <p>Conclusions</p> <p>Our results suggest that the up-regulation of Coronin-1 and Sp110, through a pathway that also includes TLR-2 up-regulation may be involved in the process of tuberculous infection in humans. However, further studies are needed, in order to elucidate whether the selective upregulation of these factors in the infected patients could serve as a specific molecular marker of tuberculosis.</p

Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures

Association Pattern of Interleukin-1 Receptor-Associated Kinase-4 Gene Polymorphisms with Allergic Rhinitis in a Han Chinese Population

Interleukin-1 receptor-associated kinase-4 (IRAK-4) encodes a kinase that is essential for NF-kB activation in Toll-like receptor and T-cell receptor signaling pathways, indicating a possible crosstalk between innate and acquired immunities. We attempted to determine whether the polymorphisms in the Interleukin-1 receptor-associated kinase-4 (IRAK-4) gene are associated with allergic rhinitis (AR) in the Han Chinese population.A population of 379 patients with AR and 333 healthy controls was studied. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE). A total of 11 single nucleotide polymorphisms (SNPs) in IRAK-4 were selected and individually genotyped.Significant allelic differences between cases and controls were obtained for the SNP of rs3794262 in the IRAK-4 gene. In the stratified analysis for gender, two SNPs (rs4251431 and rs6582484) in males appeared as significant associations. Subgroup analysis for the presence of different allergen sensitivities displayed associations only in the house dust mite-allergic cohorts (rs3794262, rs4251481). None of the selected SNPs in IRAK-4 was associated with total IgE level. The haplotype analysis indicated GCCTGCGA was significantly associated with AR. The SNP-SNP interaction information analysis indicated that the selected sets of polymorphisms had no synergistic effect.Our findings did not support the potential contribution of the IRAK-4 gene to serum IgE levels. However, the results demonstrated a gender- and allergen-dependant association pattern between polymorphisms in IRAK-4 and AR in Chinese population

LILRA2 Selectively Modulates LPS-Mediated Cytokine Production and Inhibits Phagocytosis by Monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis