Secreted and intracellular cytokines are complementary measures for human monocytes treated with Toll-like receptor agonists.

Cytokine production by human peripheral blood mononuclear cells including monocytes, is frequently assessed by measuring secreted cytokines using enzyme linked immunosorbent assay (ELISA), whereby the total concentration of one cytokine of interest is obtained without information regarding the cell type responsible for making the cytokine. Cytokines can be retained inside the cell using protein transport inhibitors. Subsequent analysis by flow cytometry not only identifies the cell type producing the cytokine but can semi-quantitate the amount of cytokine produced by measuring the geometric mean fluorescence intensity (gMFI) and is amenable to analyzing more than one protein associated with the same cell (multiplexing). We hypothesized that a more comprehensive and biologically meaningful cytokine profile could be acquired by measuring both secreted and the retained intracellular cytokines in parallel cultures of magnetic-sorted CD14+ monocytes. Peripheral monocytes were isolated from 18 healthy donors and treated with standardized molecules that stimulate cytokine production; Toll-like receptor (TLR)4 agonist (lipopolysaccharide, LPS) or TLR7/8 agonist (R848). Pro-inflammatory cytokines (interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)) secreted into the culture medium were measured by ELISA. Parallel cultures were treated with LPS and R848 in the presence of brefeldin A (protein transport inhibitor) and the accumulated intracellular cytokines measured by flow cytometry. Each cytokine (IL-6/IL-8/TNF) gave a unique general pattern when secreted versus intracellular cytokine measurements (frequency and gMFI) were plotted to determine correlation. For monocytes treated with the TLR4 agonist, secreted IL-8 correlated with the frequency of IL-8 positive cells (R = 0.559, p = .016) and not with the amount (gMFI) of IL-8 per cell. In contrast, monocytes treated with the TLR7/8 agonist showed no correlation of secreted IL-8 with the frequency of IL-8 positive cells, but with this treatment secreted IL-6 was correlated with an increase in the frequency of IL-6 positive cells (R = 0.501, p = .034). TNF secretion from monocytes treated with either the TLR4 or TLR7/8 agonist did not correlate with the frequency or gMFI of TNF positive cells. However, there were significant correlations between the TLR4 and TLR7/8 induced TNF response (secreted and gMFI). We conclude that there are fundamental differences in secreted and intracellular IL-6/IL-8/TNF production after monocytes are treated with TLR agonists. Furthermore, secreted and intracellular cytokine analyses are complementary measures that should be used in parallel to explore inflammatory response and cytokine biology.

Levels of regulatory B cells do not predict serological responses to hepatitis B vaccine.

This study investigated the immunomodulatory influence of IL10 producing B regulatory cells, Bregs (CD19+CD24hiCD38hi) to standard Twinrix® vaccination. We also investigated HBsAg specific T-cell mediated IFN-γ responses to Twinrix® which in theory could provide effective immunity despite low anti-HBs titer. A total of 309 hepatitis B negative health care students and workers completed a standard Twinrix® vaccination schedule (0, 1 and 6 months). Depending on the vaccination response the participants were divided in to non-, low- and high responders according to anti-HBs titer (<10, <100 and >1000 mIU/mL respectively) two months after completed vaccination schedule. Blood samples from baseline and after vaccination from all non- and low-responders (23 participants) and the same number of high-responders were used for flow cytometric analyses of IL10 producing Bregs and T-cell mediated IFN-γ responses. A decrease in levels of IL10 producing Bregs was observed after vaccination in high responders compared to non- and low-responders. Compiling non-and low-responders against high-responders showed a lower T-cell mediated IFN-γ response at baseline in non-and low-responders when stimulated with Engerix® vaccine. In contrary no positive correlation between IL10 producing Bregs or IFN-γ positive T-cells and anti-HBs titer was observed. Hence this study cannot prove that levels of IL10 producing Bregs or IFN-γ positive T cell affect HBV vaccine response.

High cut-off hemofiltration versus standard hemofiltration: effect on plasma cytokines.

PURPOSE: To study the effects of continuous veno-venous hemofiltration (CVVH) with high cut-off filters (CVVH-HCO) on plasma cytokine levels, sieving coefficient and clearance compared to CVVH using standard filters (CVVH-Std) in a nested cohort within a double-blind randomized controlled trial in severe acute kidney injury (AKI) patients. METHODS: We measured plasma and post-filter levels of IL-6, TNF-alpha, IL-8, IL-1 beta, RANTES, IL-10, IFN-gamma and IFN-alpha in both study groups. We also measured cytokine levels in the ultrafiltrate and calculated sieving coefficients and clearances. RESULTS: By 72 hours of treatment, IL-6 had decreased during both treatments (p = 0.009 and 0.005 respectively). In contrast, IL-10 had decreased with CVVH-Std (p = 0.03) but not CVVH-HCO (p = 0.135). None of the other cytokines showed changes over time. There were also no significant between group differences in plasma levels for each cytokine over the 72-hour treatment period. For all cytokines combined, however, the median sieving coefficient was higher for CVVH-HCO (0.31 vs. 0.16; p = 0.042) as was the mass removal rate by ultrafiltration (p = 0.027). While overall combined cytokine levels had fallen to 62.2% of baseline at 72 hours for CVVH-HCO (p<0.0001) and to 75.9% of baseline with CVVH-Std (p = 0.008) there were no between group differences. CONCLUSIONS: CVVH-HCO achieved greater combined sieving coefficient and mass removal rate by ultrafiltration for a group of key cytokines than CVVH-Std. However, this effect did not differentially lower their plasma level over the first 72 hours. Our study does not support the use of CVVH-HCO to lower cytokines in critically ill patients with AKI.

Toll-like Receptor Expression and Signaling in Peripheral Blood Mononuclear Cells Correlate With Clinical Outcomes in Acute Hepatitis C Virus Infection.

BACKGROUND: Mechanisms by which spontaneous clearance of acute hepatitis C occurs are unclear. A critical role for the innate immune system and IFNL4 polymorphisms has been proposed. This study investigates whether Toll-like receptor (TLR) expression and signaling during acute hepatitis C correlates with clinical outcomes. METHODS: Participants identified from the Australian Trial in Acute Hepatitis C and the Networks study were followed longitudinally from the time of diagnosis of acute hepatitis C. Peripheral blood mononuclear cells (PBMCs) and plasma were collected at and 2 time points after diagnosis. At each time point, TLR2, TLR4, and CD86 expression on peripheral blood monocytes, natural killer (NK) cells, and NK T cells was measured, as well as the response of PBMCs to stimulation with TLR ligands. Cytokine and chemokine levels were measured in stimulated PBMCs and plasma. RESULTS: We identified 20 participants with acute hepatitis C (10 with hepatitis C virus [HCV] monoinfection and 10 with HCV and human immunodeficiency virus coinfection). Eleven participants (55%) spontaneously cleared HCV. Acute hepatitis C and spontaneous clearance was associated with lower TLR4 expression on monocytes (P = .009) and NK cells (P = .029). Acute hepatitis C and spontaneous clearance was also associated with a reduced interferon γ response to TLR4 (P = .038) and TLR7/8 stimulation (P = .035), a reduced interleukin 6 response to TLR7/8 stimulation (P = .037), and reduced IFN-γ-inducible protein 10 (IP-10) response to TLR2 stimulation (P = .042). Lower plasma IP-10 levels were associated with spontaneous clearance (P = .001). CONCLUSIONS: These findings implicate TLR4 signaling as playing a critical role in the outcome of acute hepatitis C.

Nucleosome levels and toll-like receptor expression during high cut-off haemofiltration: a pilot assessment.

OBJECTIVES: To measure plasma nucleosome levels and expression of toll-like receptors (TLRs) in a pilot cohort of patients with severe acute kidney injury (AKI) within a randomised controlled trial of continuous venovenous haemofiltration with high cut-off filters (CVVH-HCO) v standard filters (CVVH-std). METHODS: We measured plasma nucleosome levels using the Cell Death Detection ELISA PLUS (10X) assay kit. We analysed plasma levels for correlation with disease severity and compared the effects of CVVH-HCO and CVVH-std on plasma nucleosome levels over the first 72 hours. We studied cell surface TLR expression on CD14-positive monocytes in a subcohort of CVVH-HCO patients. RESULTS: We did not detect nucleosomes in normal human plasma, but found elevated nucleosome levels in patients with severe AKI. Nucleosome levels at randomisation correlated weakly with Acute Physiology and Chronic Health Evaluation III scores (Pearson ρ=0.475, P=0.016). Treatment with CVVH-HCO or CVVH-std had no effect on nucleosome levels over 72 hours. The mean fluorescence intensity (MFI) ratios of TLR2 and TLR4 expression were elevated throughout the 72-hour period (range for TLR2, 0.97-3.98; range for TLR4, 0.91-10.18) and did not appear to decrease as a result of treatment with CVVH-HCO. CONCLUSIONS: Nucleosome concentration was elevated in the plasma of patients with severe AKI and mildly correlated with disease severity, but was not affected by treatment with CVVH-HCO or CVVH-std. Similarly, levels of TLR2 and TLR4 expression did not decrease over time during CVVHCrit HCO treatment.

Cyclosporine and tacrolimus have inhibitory effects on toll-like receptor signaling after liver transplantation.

Toll-like receptors (TLRs) play a key role in transplantation biology. The effect of immunosuppression on TLR function after liver transplantation is unknown. Peripheral blood mononuclear cells (PBMCs) from 113 post-liver transplant patients and 13 healthy controls were stimulated with TLR-specific ligands [lipopolysaccharide (TLR4), pan-3-cys (P3C) (TLR2), Poly (I:C) (PIC) (TLR3), R848 (TLR7/8), and CpG (TLR9)] for 24 hours. PBMCs from 5 healthy controls were also cultured with therapeutic concentrations of cyclosporine A (CYA) and tacrolimus (TAC). Cytokine production was measured with enzyme-linked immunosorbent assays and flow cytometry. PBMCs from patients on calcineurin inhibitors after liver transplantation produced less interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) in response to TLR2 stimulation (IL-6: P=0.02; TNFα: P=0.01), TLR4 stimulation (IL-6: P=0.02; TNFα: P=0.01), and TLR7/8 stimulation (IL-6: P=0.02; TNFα: P=0.02), compared with healthy controls. Both CD56(bright) and CD56(dim) natural killer (NK) cells from patients on calcineurin inhibitors also produced less interferon-γ (IFNγ) with TLR7/8 stimulation compared with healthy controls (CD56(bright) : P=0.002; CD56(dim) : P=0.004). Similar findings were demonstrated in healthy PBMCs cultured with CYA (PBMCs: TLR2, IL-6: P=0.005; TLR4, IL-6: P=0.03, TNFα: P=0.03; TLR7/8, IL-6: P=0.02, TNFα: P=0.01; CD56(dim) NK cells: TLR7/8, IFNγ: P=0.03). TAC impaired TLR4-mediated IL-6 and TNFα production by PBMCs (IL-6; P = 0.02; TNFα P = 0.009). In conclusion, patients on calcineurin inhibitors had impaired inflammatory cytokine production in response to TLR2, TLR4, and TLR7/8 stimulation compared comparison with healthy controls. Importantly, TAC and CYA appear to have different effects on TLR signaling. Impaired TLR function has important repercussions for risk of infection, graft rejection, and disease recurrence after transplantation, and the different immunosuppressive profiles of CYA and TAC may guide the choice of therapy to improve disease outcomes.

Vitamin D deficiency impacts on expression of toll-like receptor-2 and cytokine profile: a pilot study.

BACKGROUND: Vitamin D is believed to play an important role outside the endocrine system in the regulation of the immune system, and in cellular proliferation and differentiation. The aim of the study was to investigate the impact of vitamin D levels on innate immunity. METHODS: Participants for this prospective, longitudinal study were recruited amongst otherwise healthy staff of a large hospital in Victoria, Australia. Those fulfilling the inclusion criteria, including a vitamin D level of <50 nmol/L, were supplemented. Using flow cytometry, expression of the innate immune receptors TLR2, TLR4 and CD86 was measured on peripheral blood mononuclear cells (PBMCs) collected prior to vitamin D treatment and then at 1 and 3 months. Additonally, PBMCs at each timepoint were stimulated with specific TLR ligands and resultant supernatants were assayed for the cytokines TNFα, IL-6, IFN-α and IP-10. RESULTS: In participants whose vitamin D level was >100 nmol/L post supplementation (n=11), TLR2 expression on PBMCs increased significantly, with no change noted in TLR4 or CD86 expression. Stimulation of vitamin D deficient samples with TLR ligands produced a number of proinflammatory cytokines, which were significantly reduced upon vitamin D normalisation. In patients whose levels returned to a deficient level at 3 months despite ongoing low-level supplementation, an increase in the pro-inflamamtory state returned. This suggests that vitamin D may play an important role in ensuring an appropriate baseline pro-inflammatory state. CONCLUSIONS: This ex-vivo pilot study adds clinical evidence supporting a possibly important role for vitamin D in innate immunity. If confirmed, this unique clinical study has potentially significant implications for the treatment of a variety of inflammatory conditions, where achieving optimal vitamin D levels may help reduce inflammation.

TLR4, IL10RA, and NOD2 mutation in paediatric Crohn's disease patients: an association with Mycobacterium avium subspecies paratuberculosis and TLR4 and IL10RA expression.

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in the pathogenesis of Crohn's disease (CD). The role of CD susceptibility genes in association with these microbes is not known. Sixty-two early onset paediatric CD patients and 46 controls with known MAP status were analysed for an association with 34 single nucleotide polymorphisms (SNPs) from 18 CD susceptibility genes. Functional studies on peripheral blood mononuclear cells (PBMCs) were conducted on 17 CD patients with known CD mutations to assess IL-6, IL-10, and TNF-α expression upon stimulation with MAP precipitated protein derivative (PPD) and lipopolysaccharide (LPS). In addition, surface expression of IL10R and TLR4 on resting B cells, NK cells, T cells, and monocytes was assessed. A mutation in TLR4 (rs4986790) and IL10RA (rs22291130) was significantly associated with MAP-positive CD patients compared to MAP-negative CD patients (27.6 vs. 6.1 %, p = 0.021, and 62.1 vs. 33.3 %, p = 0.024, respectively). PPD and LPS significantly increased IL-6, IL-10, and TNF-α production in PBMCs. IL-10 and TNF-α production were significantly lower in a subgroup of CD patients (5/12) with a known NOD2 mutation. Receptor for IL-10 was significantly higher expressed on NK cells (CD56low) and on NK T cells harbouring a NOD2 mutations compared to wildtype cells (p = 0.031 and 0.005, respectively). TLR4 was significantly higher expressed on NK cells (CD56high) harbouring a NOD2 mutations compared to wildtype cells (p = 0.038).

The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like receptor signaling pathway.

BACKGROUND & AIMS: Viruses target innate immune pathways to evade host antiviral responses. Recent studies demonstrate a relationship between hepatitis B disease states and the host's innate immune response, although the mechanism of immunomodulation is unknown. In humans, the innate immune system recognizes pathogens via pattern recognition receptors such as the Toll-like receptors (TLR), initiating anti-inflammatory responses. TLR expression and pro-inflammatory cytokine production is reduced in hepatitis B e antigen (HBeAg)-positive patients following TLR stimulation. The aim of this study was to investigate interactions between TLR signaling pathways and the mature HBeAg protein localized in the cytosol. METHODS: The ability of HBeAg to inhibit TLR signaling and association with TLR adapters was evaluated by immunoprecipitation, immunostaining, and reporter studies. RESULTS: Our findings show that HBeAg co-localizes with Toll/IL-1 receptor (TIR)-containing proteins TRAM, Mal, and TLR2 at the sub-cellular level, which was not observed for Hepatitis B core antigen. Co-immunoprecipitation analysis demonstrated HBeAg interacted with TIR proteins Mal and TRAM, while a mutated HBeAg ablated interaction between Mal and MyD88. Importantly, HBeAg also disrupted homotypic TIR:TIR interaction critical for TLR-mediated signaling. Finally, HBeAg suppressed TIR-mediated activation of the inflammatory transcription factors, NF-κB and Interferon-ß promoter activity. CONCLUSIONS: Our study provides the first molecular mechanism describing HBeAg immunomodulation of innate immune signal transduction pathways via interaction and targeting of TLR-mediated signaling pathways. These finding suggest the mechanism as to how HBeAg evades innate immune responses contributing to the pathogenesis of chronic hepatitis B infection and the establishment of viral persistence.

Acute allograft rejection in human liver transplant recipients is associated with signaling through toll-like receptor 4.

BACKGROUND AND AIMS: Toll-like receptor (TLR) signaling is a crucial step in initiating adaptive immune responses. In addition to recognizing endotoxin, TLR4 also recognizes endogenous ligands ('damage-associated structures'), which are released into the circulation in the peri-transplantation period. TLR2 to a lesser extent also recognizes these endogenous ligands. Multiple studies involving solid organ transplants demonstrate a clear association between TLR4 and allograft rejection. In the present study we assessed whether an association exists between TLR4 and TLR2-dependent responses and acute liver allograft rejection. METHODS: The sample included 26 liver transplant recipients. Blood was taken pre-transplant and at multiple points over the first 14 days post-transplant. Monocytes were stimulated with TLR4 and TLR2 ligands, lipopolysaccharide and Pam-3-Cys, respectively. Monocyte TLR expression was determined using flow cytometry; enzyme-linked immunosorbent assays measured tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production. RESULTS: Nine (34.6%) patients experienced rejection. No differences existed in age, sex, disease or immunosuppression between rejectors and non-rejectors. Baseline TLR4 expression was significantly higher in rejectors (1.36 vs 1.02, P=0.01). There was no difference in TLR2 expression. In rejectors, baseline TLR4- and TLR2-dependent production of TNF-α and IL-6 was also significantly increased. Post-transplant, the two groups differed with regard to TLR4-dependent TNF-α production, with rejectors demonstrating progressive downregulation over the first week. CONCLUSIONS: Prior to liver transplantation, patients who subsequently experience rejection demonstrate robust TLR4-dependent immune responses, which are not seen in those who do not reject. This supports the theory that damage-associated structures signaling through TLR4 may be responsible for the early activation of alloimmune T-cells, favoring allograft rejection.

Effects of antibiotics on expression and function of Toll-like receptors 2 and 4 on mononuclear cells in patients with advanced cirrhosis.

BACKGROUND & AIMS: Toll-like receptors (TLRs) are critical to innate immune responses. TLR4 recognises Gram-negative bacteria, whilst TLR2 recognises Gram-positive. We examined TLR expression and function in cirrhosis, and whether this is affected by antibiotic therapy. METHODS: Sixty-four subjects were included (23 controls and 41 Child-Pugh C cirrhotic patients). Thirty patients were taking norfloxacin or trimethoprim-sulfamethoxazole as prophylaxis against bacterial peritonitis and 11 were not. In a second study, 8 patients were examined before and after commencement of antibiotics. Monocyte expression of TLR2 and 4 was determined by flow cytometry. Monocytes from the patients with paired samples were stimulated using TLR ligands and TNF-alpha production measured. RESULTS: Patients not taking antibiotics had significantly decreased TLR4 expression compared with controls (0.74 vs. 1.0, p=0.009) and patients receiving antibiotics (0.74 vs. 0.98, p=0.02). There were no differences with regard to TLR2. In the patients with paired samples, TLR4 expression increased (0.74-1.49, p=0.002) following antibiotic use, whilst again, there was no change in TLR2 expression (0.99 vs. 0.92, p=0.20). TLR4-dependent TNF-alpha production increased following antibiotic use (1077 vs. 3620pg/mL, p<0.05), whilst TLR2-dependent production was unchanged. CONCLUSIONS: TLR4 expression is decreased in patients with Child-Pugh C cirrhosis, but is restored by antibiotics targeting enteric Gram-negative bacteria. TLR4-dependent cytokine production also increases significantly following antibiotic therapy. This suggests that the high incidence of Gram-negative infection in cirrhotic patients is in part due to down-regulation of the TLR4-dependant immune response and that the efficacy of antibiotic prophylaxis is contributed to by modulation of innate immunity.

Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.

UNLABELLED: Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-alpha) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF-alpha production. CONCLUSION: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response.

Reduced expression of toll-like receptor 2 on peripheral monocytes in patients with chronic hepatitis B.

Persistent infection with hepatitis B virus (HBV) likely depends on viral inhibition of host defenses. We report that chronic hepatitis B e antigen-positive HBV infection is associated with a significant reduction in peripheral blood monocyte expression of Toll-like receptor 2, a key component of innate immunity, thereby providing a mechanism by which wild-type HBV may establish persistent infection.

B-cell antigen D8/17 is a marker of rheumatic fever susceptibility in Aboriginal Australians and can be tested in remote settings.

OBJECTIVE: To test the B-cell antigen D8/17 as a marker of past rheumatic fever (RF) in a predominantly Aboriginal Australian population, and to evaluate technical modifications to allow its use in remote settings. DESIGN AND SETTING: Cross-sectional survey in a remote Aboriginal community, a regional tertiary referral hospital and a tertiary paediatric centre in Melbourne. PARTICIPANTS: 106 people, including three with acute RF, 38 with a history of past RF, 20 relatives of these people, and 45 healthy controls. MAIN OUTCOME MEASURE: D8/17 expression in B cells. RESULTS: Blood was collected from each participant and the expression of D8/17 and CD19 in each sample was analysed by flow cytometry. The mean proportion of D8/17-positive B cells was 39.3% (SD, 11.8) in patients with previous RF, 22.5% (SD, 5.2) in first-degree relatives, 11.6% (SD, 7.2) in controls, and 83.7% (SD, 10.1) in patients with acute RF (analysis of variance test between means, P = 0.001). A cut-off of 22.1% of D8/17-positive B cells to indicate past RF yielded the highest percentage of correct results (95.4%). Delayed staining of whole blood (mean, 0.55 days; SD, 0.2) gave equivalent results to immediate staining, but the D8/17 assay on peripheral blood mononuclear cells was unreliable. CONCLUSIONS: The B-cell antigen D8/17 accurately identifies Australians with a past history of RF, and the assay is feasible in remote settings with access to facilities capable of performing D8/17 staining within half a day of sample collection.

Homologues of insecticidal toxin complex genes in Yersinia enterocolitica biotype 1A and their contribution to virulence.

Yersinia enterocolitica is an enteric pathogen that consists of six biotypes: 1A, 1B, 2, 3, 4, and 5. Strains of the latter five biotypes can carry a virulence plasmid, known as pYV, and several well-characterized chromosomally encoded virulence determinants. Y. enterocolitica strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and were once considered to be avirulent. There is growing epidemiological, clinical, and experimental evidence, however, to suggest that some biotype 1A strains are virulent and can cause gastrointestinal disease. To identify potential virulence genes of pathogenic strains of Y. enterocolitica biotype 1A, we used genomic subtractive hybridization to determine genetic differences between two biotype 1A strains: an environmental isolate, Y. enterocolitica IP2222, and a clinical isolate, Y. enterocolitica T83. Among the Y. enterocolitica T83-specific genes we identified were three, tcbA, tcaC, and tccC, that showed homology to the insecticidal toxin complex (TC) genes first discovered in Photorhabdus luminescens. The Y. enterocolitica T83 TC gene homologues were expressed by Y. enterocolitica T83 and were significantly more prevalent among clinical biotype 1A strains than other Yersinia isolates. Inactivation of the TC genes in Y. enterocolitica T83 resulted in mutants which were attenuated in the ability to colonize the gastrointestinal tracts of perorally infected mice. These results indicate that products of the TC gene complex contribute to the virulence of some strains of Y. enterocolitica biotype 1A, possibly by facilitating their persistence in vivo.

Peripheral blood mononuclear cell expression of toll-like receptors and relation to cytokine levels in cirrhosis.

Activation of macrophages by endotoxin is assumed responsible for increased circulating tumor necrosis factor alpha (TNF-alpha) and soluble TNF receptor (sTNFR) levels in cirrhosis. Relevant to this is expression of Toll-like receptor (TLR) 4 and TLR2, which is critically involved in production of TNF-alpha in response to endotoxin and Gram-positive microbial stimuli, respectively. The first studies on this in cirrhosis are reported here. In 36 cirrhotic patients and 32 controls, we measured (1) circulating endotoxin, TNF-alpha, and sTNFR levels; (2) peripheral blood mononuclear cell (PBMC) expression of TLR4 and TLR2, and (3) in vitro TNF-alpha production by PBMCs stimulated with endotoxin or Staphylococcus aureus enterotoxin B (SEB). PBMC expression of TLR2, circulating TNF-alpha levels, and in vitro TNF-alpha production were reassessed after supplementation with a synbiotic regimen known to increase intestinal levels of Gram-positive bacteria. Endotoxin, TNF-alpha, and sTNFR levels were significantly increased in cirrhosis. Endotoxin levels did not correlate significantly with other parameters. PBMC expression of TLR2 but not TLR4 was significantly up-regulated in cirrhosis and correlated significantly with serum TNF-alpha and sTNFR levels. In vitro TNF-alpha production by PBMCs stimulated by SEB was significantly blunted. Supplementation with the synbiotic regimen resulted in significant up-regulation of PBMC expression of TLR2. Serum TNF-alpha levels were further increased and in vitro TNF-alpha production further reduced in most patients. In conclusion, up-regulation of PBMC expression of TLR2 but not TLR4 occurs in cirrhosis, which implies, contrary to previous assumptions, an important stimulatory role for Gram-positive microbial components but not endotoxin. TLR2 likely contributes to increased circulating TNF-alpha and sTNFR levels in cirrhosis.