Functional miR-142a-3p Induces Apoptosis and Macrophage Polarization by Targeting tnfaip2 and glut3 in Grass Carp (Ctenopharyngodon idella).

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.

Leaky Gut Driven by Dysbiosis Augments Activation and Accumulation of Liver Macrophages via RIP3 Signaling Pathway in Autoimmune Hepatitis.

The gut-liver axis has been increasingly recognized as a major autoimmunity modulator. However, the implications of intestinal barrier in the pathogenesis of autoimmune hepatitis (AIH) remain elusive. Here, we investigated the functional role of gut barrier and intestinal microbiota for hepatic innate immune response in AIH patients and murine models. In this study, we found that AIH patients displayed increased intestinal permeability and pronounced RIP3 activation of liver macrophages. In mice models, intestinal barrier dysfunction increased intestinal bacterial translocation, thus amplifying the hepatic RIP3-mediated innate immune response. Furthermore, GSK872 dampened RIP3 activation and ameliorated the activation and accumulation of liver macrophages in vitro and in vivo experiments. Strikingly, broad-spectrum antibiotic ablation significantly alleviated RIP3 activation and liver injury, highlighting the causal role of intestinal microbiota for disease progression. Our results provided a potentially novel mechanism of immune tolerance breakage in the liver via the gut-liver axis. In addition, we also explored the therapeutic and research potentials of regulating the intestinal microbiota for the therapy of AIH.

PD-1 blockade improves Kupffer cell bacterial clearance in acute liver injury.

Patients with acute liver failure (ALF) have systemic innate immune suppression and increased susceptibility to infections. Programmed cell death 1 (PD-1) expression by macrophages has been associated with immune suppression during sepsis and cancer. We therefore examined the role of the programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) pathway in regulating Kupffer cell (KC) inflammatory and antimicrobial responses in acetaminophen-induced (APAP-induced) acute liver injury. Using intravital imaging and flow cytometry, we found impaired KC bacterial clearance and systemic bacterial dissemination in mice with liver injury. We detected increased PD-1 and PD-L1 expression in KCs and lymphocyte subsets, respectively, during injury resolution. Gene expression profiling of PD-1+ KCs revealed an immune-suppressive profile and reduced pathogen responses. Compared with WT mice, PD-1-deficient mice and anti-PD-1-treated mice with liver injury showed improved KC bacterial clearance, a reduced tissue bacterial load, and protection from sepsis. Blood samples from patients with ALF revealed enhanced PD-1 and PD-L1 expression by monocytes and lymphocytes, respectively, and that soluble PD-L1 plasma levels could predict outcomes and sepsis. PD-1 in vitro blockade restored monocyte functionality. Our study describes a role for the PD-1/PD-L1 axis in suppressing KC and monocyte antimicrobial responses after liver injury and identifies anti-PD-1 immunotherapy as a strategy to reduce infection susceptibility in ALF.

Lipopolysaccharide reduces USP13 stability through c-Jun N-terminal kinase activation in Kupffer cells.

Protein ubiquitination regulates protein stability, cellular localization, and enzyme activity. Deubiquitinases catalyze the removal of ubiquitin from target proteins and reverse ubiquitination. USP13, a deubiquitinase, has been shown to regulate a variety of cellular responses including inflammation; however, the molecular regulation of USP13 has not been demonstrated. In this study, we revealed that USP13 is degraded in response to lipopolysaccharide (LPS) in Kupffer cells. USP13 levels are significantly decreased in inflamed organs, including liver tissues from septic mice. LPS reduces USP13 protein stability, not transcription, in Kupffer cells. Furthermore, LPS increases USP13 polyubiquitination. Inhibition of proteasome, but not lysosome or immunoproteasome, attenuates LPS-induced USP13 degradation, suggesting USP13 degradation is mediated by the ubiquitin-proteasome system. A catalytically inactive form of USP13 exhibits similar degree of degradation compared with USP13 wild-type, suggesting that USP13 degradation is not dependent on its activity. Furthermore, USP13 degradation is dependent on new protein synthesis. Inhibition of c-Jun N-terminal kinase (JNK) attenuates USP13 degradation, indicating that JNK-dependent new protein synthesis is necessary for USP13 degradation. This study reveals a molecular mechanism of regulation of USP13 degradation in Kupffer cells in response to bacterial endotoxin.

The surreptitious survival of the emerging pathogen Staphylococcus lugdunensis within macrophages as an immune evasion strategy.

Staphylococcus lugdunensis is a commensal bacterium that can cause serious infection suggesting an ability to circumvent aspects of host immunity. We demonstrate here that macrophages fail to kill ingested S. lugdunensis and the bacteria persist for extended periods, without replicating, within mature LAMP-1-positive phagolysosomes. Phagocytosed S. lugdunensis also do not intoxicate host cells in contrast to Staphylococcus aureus. Optimal survival of S. lugdunensis requires O-acetylated peptidoglycan because an oatA mutant, which is more sensitive to killing by lysozyme than wild type, survived to a lesser extent in macrophages. In vitro models of macrophage infection reveal that viable intracellular S. lugdunensis bacteria can be made to grow by pharmacologic perturbation of phagosome function or by phagocyte intoxication by S. aureus toxins. Remarkably, replicating S. lugdunensis is not constrained by LAMP-1 and phosphatidylserine-positive endomembranes, which is distinct from S. aureus that replicates within phagolysosomes. In vivo, S. lugdunensis can also reside in the murine Kupffer cell where the bacteria persist without replicating and require oatA to resist killing in vivo. The intracellular environment of the macrophage represents a niche where S. lugdunensis can exist while protected from extracellular immune factors and may serve as a reservoir from which these bacteria could disseminate.

A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.

The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

Contribution of programmed cell death receptor (PD)-1 to Kupffer cell dysfunction in murine polymicrobial sepsis.

Recent studies suggest that coinhibitory receptors appear to be important in contributing sepsis-induced immunosuppression. Our laboratory reported that mice deficient in programmed cell death receptor (PD)-1 have increased bacterial clearance and improved survival in experimental sepsis induced by cecal ligation and puncture (CLP). In response to infection, the liver clears the blood of bacteria and produces cytokines. Kupffer cells, the resident macrophages in the liver, are strategically situated to perform the above functions. However, it is not known if PD-1 expression on Kupffer cells is altered by septic stimuli, let alone if PD-1 ligation contributes to the altered microbial handling seen. Here we report that PD-1 is significantly upregulated on Kupffer cells during sepsis. PD-1-deficient septic mouse Kupffer cells displayed markedly enhanced phagocytosis and restoration of the expression of major histocompatibility complex II and CD86, but reduced CD80 expression compared with septic wild-type (WT) mouse Kupffer cells. In response to ex vivo LPS stimulation, the cytokine productive capacity of Kupffer cells derived from PD-1-/- CLP mice exhibited a marked, albeit partial, restoration of the release of IL-6, IL-12, IL-1ß, monocyte chemoattractant protein-1, and IL-10 compared with septic WT mouse Kupffer cells. In addition, PD-1 gene deficiency decreased LPS-induced apoptosis of septic Kupffer cells, as indicated by decreased levels of cleaved caspase-3 and reduced terminal deoxynucleotidyl transferase dUTP nick end-labeling-positive cells. Exploring the signal pathways involved, we found that, after ex vivo LPS stimulation, septic PD-1-/- mouse Kupffer cells exhibited an increased Akt phosphorylation and a reduced p38 phosphorylation compared with septic WT mouse Kupffer cells. Together, these results indicate that PD-1 appears to play an important role in regulating the development of Kupffer cell dysfunction seen in sepsis.

Lactobacillus paracasei Induces M2-Dominant Kupffer Cell Polarization in a Mouse Model of Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Gut microbiota may be associated with the pathogenesis of nonalcoholic steatohepatitis (NASH). This study aimed to investigate the protective effects and possible mechanisms of Lactobacillus paracasei on NASH. METHODS: Thirty male C57BL/6 mice were randomized into three groups and maintained for 10 weeks: control group (standard chow), NASH model group (high fat + 10 % fructose diet), and the L. paracasei group (NASH model with L. paracasei). Liver histology, serum aminotransferase levels, and hepatic gene expression levels were measured. Intestinal permeability was investigated using urinary (51)Creatinine Ethylenediaminetetraacetic acid ((51)Cr-EDTA) clearance. Total Kupffer cell counts and their composition (M1 vs. M2 Kupffer cells) were measured using flow cytometry with F4/80 and CD206 antibodies. RESULTS: Hepatic fat deposition, serum ALT level, and (51)Cr-EDTA clearance were significantly lower in the L. paracasei group than the NASH group (p < 0.05). The L. paracasei group had lower expression in Toll-like receptor-4 (TLR-4), NADPH oxidase-4 (NOX-4), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin 4 (IL-4), peroxisome proliferator activated receptor gamma (PPAR-γ), and PPAR-δ compared with the NASH group (p < 0.05). The total number of F4/80(+) Kupffer cells was lower in the L. paracasei group than the NASH group. L. paracasei induced the fraction of F4/80(+)CD206(+) cells (M2 Kupffer cells) while F4/80(+)CD206(-) cells (M1 Kupffer cells) were higher in the NASH group (F4/80(+)CD206(+) cell: 44% in NASH model group vs. 62% in L. paracasei group, p < 0.05). CONCLUSIONS: Lactobacillus paracasei attenuates hepatic steatosis with M2-dominant Kupffer cell polarization in a NASH model.

Infection as a Trigger for Portal Hypertension.

BACKGROUND: Microbial infections are a relevant problem for patients with liver cirrhosis. Different types of bacteria are responsible for different kinds of infections: Escherichia coli and Klebsiella pneumoniae are frequently observed in spontaneous bacterial peritonitis or urinary tract infections, and Streptococcus pneumoniae and Mycoplasma pneumoniae in pulmonary infections. Mortality is up to 4-fold higher in infected patients with liver cirrhosis than in patients without infections. Key Messages: Infections in patients with liver cirrhosis are due to three major reasons: bacterial translocation, immune deficiency and an increased incidence of systemic infections. Nonparenchymal liver cells like Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells are the first liver cells to come into contact with microbial products when systemic infection or bacterial translocation occurs. Kupffer cell (KC) activation by Toll-like receptor (TLR) agonists and endothelial sinusoidal dysfunction have been shown to be important mechanisms increasing portal pressure following intraperitoneal lipopolysaccharide pretreatment in cirrhotic rat livers. Reduced intrahepatic vasodilation and increased intrahepatic vasoconstriction are the relevant pathophysiological pathways. Thromboxane A2 and leukotriene (LT) C4/D4 have been identified as important vasoconstrictors. Accordingly, treatment with montelukast to inhibit the cysteinyl-LT1 receptor reduced portal pressure in cirrhotic rat livers. Clinical studies have demonstrated that activation of KCs, estimated by the amount of soluble CD163 in the blood, correlates with the risk for variceal bleeding. Additionally, intestinal decontamination with rifaximin in patients with alcohol-associated liver cirrhosis reduced the portal pressure and the risk for variceal bleeding. CONCLUSIONS: TLR activation of nonparenchymal liver cells by pathogens results in portal hypertension. This might explain the pathophysiologic correlation between microbial infections and portal hypertension in patients with liver cirrhosis. These findings are the basis for both better risk stratifying and new treatment options, such as specific inhibition of TLR for patients with liver cirrhosis and portal hypertension.

CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver.

CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/-) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

Host response to translocated microbial products predicts outcomes of patients with HBV or HCV infection.

BACKGROUND & AIMS: Chronic infection with hepatitis B or C virus (HBV or HCV) is a leading cause of cirrhosis by unknown mechanisms of pathogenesis. Translocation of gut microbial products into the systemic circulation might increase because of increased intestinal permeability, bacterial overgrowth, or impaired clearance of microbial products by Kupffer cells. We investigated whether the extent and progression of liver disease in patients with chronic HBV or HCV infection are associated with microbial translocation and subsequent activation of monocytes. METHODS: In a retrospective study, we analyzed data from 16 patients with minimal fibrosis, 68 with cirrhosis, and 67 uninfected volunteers. We analyzed plasma levels of soluble CD14 (sCD14), intestinal fatty acid binding protein, and interleukin-6 by enzyme-linked immunosorbent assay, and lipopolysaccharide (LPS) by the limulus amebocyte lysate assay, at presentation and after antiviral treatment. RESULTS: Compared with uninfected individuals, HCV- and HBV-infected individuals had higher plasma levels of LPS, intestinal fatty acid binding protein (indicating enterocyte death), sCD14 (produced upon LPS activation of monocytes), and interleukin-6. Portal hypertension, indicated by low platelet counts, was associated with enterocyte death (P=.045 at presentation, P<.0001 after therapy). Levels of sCD14 correlated with markers of hepatic inflammation (P=.02 for aspartate aminotransferase, P=.002 for ferritin) and fibrosis (P<.0001 for γ-glutamyl transpeptidase, P=.01 for alkaline phosphatase, P<.0001 for α-fetoprotein). Compared to subjects with minimal fibrosis, subjects with severe fibrosis at presentation had higher plasma levels of sCD14 (P=.01) and more hepatic CD14+ cells (P=.0002); each increased risk for disease progression (P=.0009 and P=.005, respectively). CONCLUSIONS: LPS-induced local and systemic inflammation is associated with cirrhosis and predicts progression to end-stage liver disease in patients with HBV or HCV infection.

Cardiac dysfunction induced by experimental myocardial infarction impairs the host defense response to bacterial infection in mice because of reduced phagocytosis of Kupffer cells.

OBJECTIVE: This study was undertaken to investigate the effects of cardiac dysfunction induced by experimental myocardial infarction on the host defense response to bacterial infection and the role of Kupffer cells in mediating this response. METHODS: Myocardial infarction was induced in C57BL/6 mice by ligation of the left anterior descending coronary artery. Mice were challenged with Escherichia coli intravenously 1, 5, and 14 days after myocardial infarction or sham operation. Thereafter, the cytokine production and the function of their Kupffer cells were assessed. RESULTS: Mice with myocardial infarction showed remarkable cardiac dysfunction and had a significantly lower survival than sham mice after bacterial challenge at 5 days after surgery; bacterial challenge at 1 or 14 days after surgery resulted in no difference in survival between myocardial infarction and sham mice. The phagocytic activity of Kupffer cells, assessed by fluorescein isothiocyanate microspheres, remarkably decreased in mice with myocardial infarction 5 days after surgery. Serum peaks of tumor necrosis factor and interferon-gamma after bacterial challenge were also suppressed in mice with myocardial infarction at 5 days. Production of these cytokines and immunoglobulin-M from liver mononuclear cells was also impaired in mice with myocardial infarction. Enhancement of the phagocytic activity of Kupffer cells by C-reactive protein significantly improved survival after infection in mice with myocardial infarction, although neither interleukin-18 nor immunoglobulin-M treatment improved survival. CONCLUSIONS: Cardiac dysfunction induced by myocardial infarction renders mice susceptible to bacterial infection and increases mortality because of a reduced ability of Kupffer cells to clear infectious bacteria. C-reactive protein-enhanced phagocytic activity of Kupffer cells may improve the poor prognosis after bacterial infection in mice with myocardial infarction.

"The very pulse of the machine": the tuberculous granuloma in motion.

What is happening inside the tuberculous granuloma? In this issue of Immunity, Egen et al. (2008) present live images of tuberculous granulomas of the mouse, demonstrating the influx and incessant wandering of T lymphocytes.

Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.

Granulomas play a key role in host protection against mycobacterial pathogens, with their breakdown contributing to exacerbated disease. To better understand the initiation and maintenance of these structures, we employed both high-resolution multiplex static imaging and intravital multiphoton microscopy of Mycobacterium bovis BCG-induced liver granulomas. We found that Kupffer cells directly capture blood-borne bacteria and subsequently nucleate formation of a nascent granuloma by recruiting both uninfected liver-resident macrophages and blood-derived monocytes. Within the mature granuloma, these myeloid cell populations formed a relatively immobile cellular matrix that interacted with a highly dynamic effector T cell population. The efficient recruitment of these T cells was highly dependent on TNF-alpha-derived signals, which also maintained the granuloma structure through preferential effects on uninfected macrophage populations. By characterizing the migration of both innate and adaptive immune cells throughout the process of granuloma development, these studies provide a new perspective on the cellular events involved in mycobacterial containment and escape.

Pseudomonas aeruginosa delays Kupffer cell death via stabilization of the X-chromosome-linked inhibitor of apoptosis protein.

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.

Lipid peroxidation and serum cytokines in acute alcoholic hepatitis.

AIMS: Increased exposure of Kupffer cells to intestinal-borne Gram-negative bacteria enhances the metabolism and leads to cytokine production by these cells. Activation of Kupffer cells increases free radical release, which may, in turn, enhance cytokine secretion, creating a positive feedback loop, which contributes to liver inflammation. Cytokines act on T cells, inducing their proliferation and secretion of additional interleukins. Lipid peroxidation products (malondialdehyde; MDA) form adducts with proteins and acetaldehyde, triggering a T cell immune response. Controversy exists about the predominance of either Th-1 or Th-2 cellular responses. We performed the present study in order to analyse the cytokine pattern in patients with acute alcoholic hepatitis, its relation to MDA and the relation between all these parameters and liver function and prognosis. SUBJECTS AND METHODS: The study included 53 male alcoholics, 47 followed up for a median time of 32 months, during which 17 of them died. We measured serum MDA, tumour necrosis factor-alpha, interferon gamma (IFNG) and interleukins (IL) 4, 6, 8, and 10. RESULTS: MDA levels were raised in cirrhotics and non-cirrhotics with alcoholic hepatitis, maintaining a relationship with bilirubin and Maddrey index, and with mortality in the univariate analysis. Both IFNG and IL-4 were raised in our patients compared with controls, as well as IL-8, and IL-6, but IL-10 were below the detection limit in the majority of cases, especially in cirrhotics. Using a Cox regression model, Maddrey index displaced MDA in the survival analysis. CONCLUSIONS: Our data lend support to the hypothesis that activation of both Th-1 and Th-2 cell subsets take place. MDA levels are raised in alcoholics with alcoholic hepatitis and are closely related to liver function derangement and to survival, although this is displaced by Maddrey index using Cox regression model.

Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection.

AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C. pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes. METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-alpha by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzyme-linked immunosorbent assay. RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20. The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture. Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-alpha levels. CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis.

Inactivation of Kupffer cells by gadolinium administration prevents lipopolysaccharide-induced decrease in liver insulin-like growth factor-I and IGF-binding protein-3 gene expression.

Gram-negative bacterial infection or treatment of animals with bacterial lipopolysaccharide (LPS) induces a catabolic state with proteolysis, liver injury and an inhibition of the insulin-like growth factor-I (IGF-I) system. The purpose of this work was to elucidate the role of Kupffer cells in LPS-induced inhibition of the IGF-I/IGF-binding protein-3 (IGFBP-3) system. Adult male Wistar rats were either pretreated with the Kupffer cell inhibitor gadolinium chloride (10 mg/kg, i.v., 24 h prior to LPS exposure) or saline vehicle. Rats received two i.p. injections of 1 mg/kg LPS (at 17:30 and 08:30 h the following day) and were killed 4 h after the second injection. LPS administration induced a significant decrease in body weight and in serum concentrations of IGF-I and IGFBP-3 (P < 0.01), as well as in their gene expression in the liver. LPS-injected rats had increased serum concentrations of ACTH, corticosterone (P < 0.05), tumour necrosis factor-alpha (TNF-alpha) and nitrites (P < 0.01). Pretreatment of the animals with gadolinium chloride blocked the inhibitory effect of LPS on body weight, and on serum concentrations of IGF-I, IGFBP-3 and nitrites, as well as growth hormone receptor (GHR), IGF-I and IGFBP-3 gene expression in the liver. In contrast, gadolinium chloride administration did not modify the stimulatory effect of LPS on serum concentrations of ACTH, corticosterone and TNF-alpha. These results suggest that Kupffer cells are important mediators in the inhibitory effect of LPS on GHR, IGF-I and IGFBP-3 gene expression in the liver, leading to a decrease in serum concentrations of IGF-I and IGFBP-3.

Cytokine responses to fungal pathogens in Kupffer Cells are Toll-like receptor 4 independent and mediated by tyrosine kinases.

Disseminated fungal infections are increasing. However, the interactions between the body's largest population of tissue macrophages, the Kupffer cells and the fungal pathogens are scarcely understood. The aim of this study was to examine the involvement of Toll-like receptor 4 (TLR4) signalling in cytokine production, using primary cultures of rat and murine Kupffer cells exposed to Aspergillus fumigatus and Candida albicans hyphae and conidia. All fungal components induced the release of tumour necrosis factor-alpha (TNF-alpha), but with delayed kinetics compared with lipopolysaccharide (LPS). Candida albicans was the most potent inducer of TNF-alpha protein and mRNA and the only inducer of interleukin-10 (IL-10) in rat Kupffer cells. All fungal components induced enhanced mRNA levels of macrophage inhibitory protein-2 (MIP-2) in the cells, similar to LPS. Inhibitors of Src tyrosine kinases added to cells prior to stimulation led to attenuation in the release of both TNF-alpha (60%, P < 0.05) and IL-10 (70%, P < 0.05) induced by C. albicans conidia but did not influence the LPS-mediated cytokine release. Murine Kupffer cells (C57BL/10J) also released TNF-alpha as well as the chemokines keratinocyte-derived chemokine (KC) and MIP-2 in response to fungal component. Surprisingly, Kupffer cells from TLR4-deficient C57BL/ScCr mice exhibited significantly enhanced production of KC and MIP-2 upon stimulation by fungal components compared with control littermates (P < 0.05). Our study demonstrates that Aspergillus and Candida components induce cytokine production in rat Kupffer cells and that the response to C. albicans conidia involves Src tyrosine kinases. The experiments with TLR4-deficient Kupffer cells suggest that the cytokine response in these cells to fungal component is not mediated by TLR4.