Genetic Variation/Evolution and Differential Host Responses Resulting from In-Patient Adaptation of Mycobacterium avium.

Members of the Mycobacterium avium complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome. The current study aimed to characterize the genomic changes within M.avium isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Isolates from patients (patients 4, 9, and 13) for whom more than two isolates were available were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single-nucleotide polymorphisms (SNPs), suggesting that M. avium accumulates mutations at higher rates during persistent infections than other mycobacteria. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency toward increased persistence and the downregulation of inflammatory cytokines by host-adapted M. avium strains. The study revealed the rapid genetic evolution of M. avium in chronically infected patients, accompanied by changes in the virulence properties of the sequential mycobacterial isolates.

Expression of Toll-like receptor-3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2.

Anti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti-microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor (TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin (IL)-1ß and the TLR-3 ligand poly(I:C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.

The association between neutrophil gelatinase-associated lipocalin and clinical outcome in chronic heart failure: results from CORONA*.

OBJECTIVE: To study the prognostic value of neutrophil gelatinase-associated lipocalin (NGAL) in chronic heart failure (HF) of ischaemic aetiology. BACKGROUND: Neutrophil gelatinase-associated lipocalin is a marker of kidney injury as well as matrix degradation and inflammation and has previously been shown to be increased in HF. We investigated whether serum NGAL levels could provide prognostic information in chronic HF. METHODS: We assessed NGAL as a predictor of primary outcomes (cardiovascular death, nonfatal stroke and nonfatal myocardial infarction, n = 307) and all-cause mortality (n = 321), cardiovascular mortality (n = 259) and hospitalization (n = 647) as well as the number of hospitalizations during follow-up for all (n = 1934) and CV causes (n = 1204) in 1415 patients with chronic HF (≥60 years, New York Heart Association class II-IV, ischaemic systolic HF) in the CORONA population, randomly assigned to 10 mg rosuvastatin or placebo. Results. Multivariate analysis revealed that NGAL added significant information when adjusting for clinical variables, but was no longer significant when further adjusting for apolipoprotein A-1 (ApoA-1), glomerular filtration rate (GFR), C-reactive protein (CRP) and N-terminal pro-brain natriuretic peptide (NT-proBNP). However, belonging to the highest NGAL tertile was associated with more frequent hospitalization, even after adjusting for clinical variables, GFR and ApoA-1, but not after adjusting for CRP and NT-proBNP. There was no interaction between rosuvastatin treatment and NGAL. Conclusion. Neutrophil gelatinase-associated lipocalin added no significant information to NT-proBNP and GFR in a multivariate model for primary and secondary end-points.

Enhanced levels of CCL19 in patients with advanced acquired immune deficiency syndrome (AIDS).

Based on the ability to recruit lymphocytes and dendritic cells to lymphoid tissue and to promote inflammation, we hypothesized a role for dysregulated CCL19 and CCL21 levels in human immunodeficiency virus (HIV)-infected patients with advanced immunodeficiency, and in particular in those with accompanying Mycobacterium avium complex (MAC) infection. The hypothesis was explored by studies in HIV-infected patients with and without MAC infection, as well as in vitro, examining the ability of proteins from MAC to promote CCL19 and CCL21 responses in peripheral blood mononuclear cells (PBMC) during highly active anti-retroviral therapy (HAART). Our main findings were: (i) raised serum levels of CCL19 in HIV-infected patients with CD4(+) T cell count <50 cells/µl compared with HIV-infected patients with CD4(+) T cell count >500 cells/µl and healthy controls, with particularly high levels in those with MAC infection; (ii) elevated plasma levels of CCL19 predicted a higher mortality in acquired immune deficiency syndrome (AIDS)-patients, independent of ongoing MAC infection; and (iii) marked production of CCL19 in MAC-stimulated peripheral blood mononuclear cells (PBMC) and pronounced disturbances in MAC-induced CCL19 production in PBMC from HIV patients that was partly reversed during HAART. Our findings suggest the involvement of CCL19 in AIDS patients with advanced immunodeficiency, potentially mediating both adaptive and maladaptive responses.

Decreased serum lipocalin-2 levels in human immunodeficiency virus-infected patients: increase during highly active anti-retroviral therapy.

Although neutrophil gelatinase-associated lipocalin (NGAL) may play a pivotal role in the innate immune response, there are currently no data on NGAL levels in human immunodeficiency virus (HIV)-infected patients. In this study we aimed to examine the regulation of NGAL in HIV infection. The regulation of NGAL in HIV infection was examined by different experimental approaches, including studies in peripheral blood and mononuclear cells (MNC) from bone marrow aspirates before and during highly active anti-retroviral therapy (HAART). We found that: before initiating HAART, HIV-infected patients (n = 37) had significantly decreased serum NGAL levels compared with healthy controls (n = 26); (ii) during HAART, there was a gradual and significant increase in NGAL concentrations reaching levels comparable to those in healthy controls after 12 months; (iii) this increase was seen primarily in virological responders to HAART (HIV RNA level <200 copies/ml after 24 months); (iv) phytohaemagglutinin-stimulated NGAL release in MNC cells from bone marrow aspirates was decreased in untreated HIV-infected patients compared with healthy controls, but increased after 26 weeks on HAART; and (v) there was a significant positive correlation between neutrophil counts and NGAL levels at all time-points during HAART. We have shown decreased NGAL levels in HIV-infected patients, potentially reflecting decreased number and function of neutrophils as well as impaired bone marrow myelopoiesis. These abnormalities were reversed by successful HAART. Our findings underscore further the involvement of neutrophils and innate immunity in HIV-related immunodeficiency.

Differential expression of Toll-like receptor 2 in human cells.

Human Toll-like receptor 2 (TLR2) is a receptor for a variety of microbial products and mediates activation signals in cells of the innate immune system. We have investigated expression and regulation of the TLR2 protein in human blood cells and tissues by using two anti-TLR2 mAbs. Only myelomonocytic cell lines expressed surface TLR2. In tonsils, lymph nodes, and appendices, activated B-cells in germinal centers expressed TLR2. In human blood, CD14+ monocytes expressed the highest level of TLR2 followed by CD15+ granulocytes, and CD19+ B-cells, CD3+ T-cells, and CD56+ NK cells did not express TLR2. The level of TLR2 on monocytes was after 20 h up-regulated by LPS, GM-CSF, IL-1, and IL-10 and down-regulated by IL-4, IFN-gamma, and TNF. On purified granulocytes, LPS, GM-CSF, and TNF down-regulated, and IL-10 modestly increased TLR2 expression after 2 h. These data suggest that TLR2 protein expression in innate immune cells is differentially regulated by inflammatory mediators.

Human toll-like receptor 2 mediates monocyte activation by Listeria monocytogenes, but not by group B streptococci or lipopolysaccharide.

Human Toll like receptor (TLR) 2 has been implicated as a signaling receptor for LPS from Gram-negative bacteria and cell wall components from Gram-positive organisms. In this study, we investigated whether TLR2 can signal cell activation by the heat-killed group B streptococci type III (GBS) and Listeria monocytogenes (HKLM). HKLM, but not GBS, showed a time- and dose-dependent activation of Chinese hamster ovary cells transfected with human TLR2, as measured by translocation of NF-kappaB and induction of IL-6 production. A mAb recognizing a TLR2-associated epitope (TL2.1) was generated that inhibited IL-6 production from Chinese hamster ovary-TLR2 cells stimulated with HKLM or LPS. The TL2.1 mAb reduced HKLM-induced TNF production from human monocytes by 60%, whereas a CD14 mAb (3C10) reduced the TNF production by 30%. However, coadministrating TL2.1 and 3C10 inhibited the TNF response by 80%. In contrast to this, anti-CD14 blocked LPS-induced TNF production from monocytes, whereas anti-TLR2 showed no inhibition. Neither TL2.1 nor 3C10 affected GBS-induced TNF production. These results show that TLR2 can function as a signaling receptor for HKLM, possibly together with CD14, but that TLR2 is unlikely to be involved in cell activation by GBS. Furthermore, although LPS can activate transfected cell lines through TLR2, this receptor does not seem to be the main transducer of LPS activation of human monocytes. Thus, our data demonstrate the ability of TLR2 to distinguish between different pathogens.

The tumor necrosis factor-inducing potency of lipopolysaccharide and uronic acid polymers is increased when they are covalently linked to particles.

Lipopolysaccharide (LPS) and polymers of the uronic acid family stimulate monocytes to produce tumor necrosis factor (TNF). The TNF-inducing potency of these polysaccharides may depend on their supramolecular configuration. In this study detoxified LPS and uronic acid polymers have been covalently linked to particles which have been added to monocytes under serum-free conditions. Reducing the size of mannuronan from 350,000 to 5,500 Da (M-blocks) led to a 10- to 100-fold reduction in TNF-inducing potency. However, covalently linking the M-blocks to monodisperse suspensions of magnetic particles increased the TNF-inducing potency by up to 60,000-fold. Also, the TNF-inducing potency of glucuronic acid polymers was increased when they were linked to particles, but no potentiation was observed with guluronic acid blocks covalently attached to particles. Furthermore, O chains of LPS (detoxified LPS) became potent TNF inducers when they were presented to monocytes on a particle surface. No activation of the LPS-responsive SW480 adenocarcinoma cells was found with detoxified LPS or M-block particles, suggesting a preference for cells expressing CD14 and/or other membrane molecules. The potentiating effects were not restricted to polymers attached to aminated magnetic particles. Of particular interest, we found that short blocks of mannuronan induced TNF production also when covalently linked to biodegradable, bovine serum albumin particles.

Human monocyte receptors involved in tumor necrosis factor responses to group B streptococcal products.

Several group B streptococcal products have been previously found to stimulate human monocytes to produce tumor necrosis factor alpha. In order to identify the receptors involved in these responses, monocytes were stimulated with purified group- or type-specific carbohydrates or lipoteichoic acid in the presence of anti-receptor monoclonal antibodies, soluble CD14, or lipopolysaccharide-binding protein. Results indicate that CD14 plays an important role in tumor necrosis factor alpha responses to all of the stimuli tested. Moreover, both CD14 and complement receptor type 3 may be involved in responses to the group-antigen.

Beta 2 integrins are involved in cytokine responses to whole Gram-positive bacteria.

Proinflammatory cytokines have an important pathophysiologic role in septic shock. CD14 is involved in cytokine responses to a number of purified bacterial products, including LPS. However, little is known of monocyte receptors involved in cytokine responses to whole bacteria. To identify these receptors, human monocytes were pretreated with different mAbs and TNF-alpha was measured in culture supernatants after stimulation with whole heat-killed bacteria. Human serum and anti-CD14 Abs significantly increased and decreased, respectively, TNF-alpha responses to the Gram-negative Escherichia coli. However, neither treatment influenced responses to any of the Gram-positive bacteria tested, including group A and B streptococci, Listeria monocytogenes, and Staphylococcus aureus. Complement receptor type III (CR3 or CD18/CD11b) Abs prevented TNF-alpha release induced by heat-killed group A or B streptococci. In contrast, the same Abs had no effects when monocytes were stimulated with L. monocytogenes or S. aureus. Using either of the latter bacteria, significant inhibition of TNF-alpha release was produced by Abs to CD11c, one of the subunits of CR4. To confirm these blocking Ab data, IL-6 release was measured in CR3-, CR4-, or CD14-transfected Chinese hamster ovary cells after bacterial stimulation. Accordingly, streptococci triggered moderate IL-6 production (p < 0.05) in CR3 but not CD14 or CR4 transfectants. In contrast, L. monocytogenes and S. aureus induced IL-6 release in CR4 but not CR3 or CD14 transfectants. Collectively our data indicate that beta 2 integrins, such as CR3 and CR4, may be involved in cytokine responses to Gram-positive bacteria. Moreover, CD14 may play a more important role in responses to whole Gram-negative bacteria relative to Gram-positive ones.

Involvement of CD14 and toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae.

Invasive fungal infections represent an increasing problem associated with high mortality. The present study was undertaken to identify leukocyte subsets that are activated by hyphal fragments in a whole-human-blood model, as well as to examine the involvement of CD14 and Toll-like receptors (TLRs) in activation of monocytes by hyphae. Incubation of whole human blood with hyphal fragments from Aspergillus fumigatus and Scedosporium prolificans for 6 h caused induction of mRNAs for tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in T cells, B cells, and monocytes, but not in granulocytes, as analyzed by reverse transcription-PCR with mRNA isolated from very pure populations of these leukocyte subsets. In primary adherent human monocytes, induction of TNF-alpha by hyphal fragments was dependent on plasma. Heat treatment of plasma at 56 degrees C for 30 min strongly reduced the ability of plasma to prime for activation. Pretreatment of human monocytes with different concentrations (1, 3, and 10 microg/ml) of monoclonal antibody (MAb) HTA125 (anti-TLR4) or MAb 18D11 (anti-CD14) for 30 min inhibited the release of TNF-alpha induced by hyphal fragments in a dose-dependent manner. Maximal inhibitions of 35 and 70% were obtained with 10 microg of HTA125 and 18D11 per ml, respectively. In contrast, pretreatment with MAb TL2.1 (anti-TLR2) did not affect signaling induced by hyphae. Pretreatment with the lipid A antagonist B975 blocked lipopolysaccharide signaling but did not inhibit TNF-alpha production induced by hyphal fragments. Our results suggest that T cells, B cells, and monocytes are involved in the innate immune response to invasive fungal pathogens and that serum components are relevant for activation of monocytes by hyphae. CD14 and TLR4 may be involved in signaling of Aspergillus hyphae in monocytes, but further studies to elucidate this issue are warranted.

Involvement of CD14 and beta2-integrins in activating cells with soluble and particulate lipopolysaccharides and mannuronic acid polymers.

Lipopolysaccharide (LPS) and related bacterial products can be recognized by host inflammatory cells in a particulate, bacterium-bound form, as well as in various soluble, released forms. In the present study we have compared the mechanisms used by LPS, detoxified LPS (DLPS), and mannuronic acid polymers (M-polymers), in solution or covalently linked to particles, in stimulating monocytes to tumor necrosis factor (TNF) production. The addition of recombinant LPS binding protein (LBP) and/or soluble CD14 (sCD14) enhanced the production of TNF from monocytes stimulated with soluble LPS, DLPS, or M-polymer, but did not affect the response to M-polymer or DLPS attached to particles. Treatment of monocytes with antibody to CD14, CD18, or CD11b showed that CD14, but not CR3 (CD11b/CD18), mediated monocyte TNF production in response to the soluble antigens. In contrast, anti-CD14, anti-CD11b and anti-CD18 monoclonal antibodies all inhibited the response to the particulate stimuli. On the other hand, B975, a synthetic analog of Rhodobacter capsulatus lipid A, completely abrogated the monocyte TNF response induced by LPS but did not affect the TNF induction by DLPS or M-polymer, either in soluble or particulate forms. These data demonstrate that the engagement of immune receptors by bacterial products such as LPS, DLPS, and M-polymer is dependent upon the presentation form of their constituent carbohydrates, and that factors such as aggregation state, acylation, carbohydrate chain length, and solid versus liquid phase of bacterial ligands influence the mechanisms used by cells in mediating proinflammatory responses.

Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.

Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion.