Protective effect of OK-432 on mice against endotoxemia and infection with Pseudomonas aeruginosa and Salmonella enteritidis

OK-432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK-432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pretreatment of OK-432 reduced the lethality of lipopolysaccharide (LPS)-induced endotoxic shock in D-(+)-galactosamine-sensitized C3H/HeN mice. OK-432 did not affect the TNFalpha production in blood, but it did decrease the susceptibility to TNFalpha. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK-432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK-432 against the endotoxemia and bacteremia may depend on an up-regulation of clearance of LPS and bacteria and the augmented resistance to TNFalpha.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

Apolipoprotein E knock-out mice are highly susceptible to endotoxemia and Klebsiella pneumoniae infection

Lipoproteins are able to neutralize bacterial lipopolysaccharide (LPS) and thereby inhibit the proinflammatory cytokine response. In a previous study, we demonstrated that hypercholesterolemic low density lipoprotein receptor knock-out (LDLr-/-) mice are protected against lethal endotoxemia and gram-negative infection. In the present study we investigated the susceptibility of apolipoprotein E knock-out mice (apoE-/-) to LPS and to Klebsiella pneumoniae. These mice have increased plasma lipoprotein concentrations in the very low density lipoprotein (VLDL)-sized fraction. Despite 8 -fold higher plasma cholesterol levels compared to controls, and in contrast to LDLr-/- mice, apoE-/- mice were significantly more susceptible to endotoxemia and to K. pneumoniae infection. Circulating TNFalpha concentrations after intravenously injected LPS were 4 - to 5-fold higher in apoE-/- mice, whereas IL-1alpha, IL-1beta, and IL-6 did not differ. This TNF response was not due to an increased cytokine production capacity of cells from apoE-/- mice, as ex vivo cytokine production in response to LPS did not differ between apoE-/- and control mice. The LPS-neutralizing capacity of apoE-/- plasma was significantly less than that of controls. Most likely, the absence of apoE itself in the knock-out mice explains the failure to neutralize LPS, despite the very high cholesterol concentrations.     

The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo

We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.     

Effects of IL-18 and IL-10 pre-treatment on the alteration of endogenous cytokines in liver and spleen of mice with experimental endotoxemia

Mechanisms of interleukin-18 (IL-18) and interleukin-10 (IL-10) in lipopolysaccharide (LPS) induced endotoxemia are not clear; their protective role is being investigated so that they may effectively modulate the host cytokine levels during endotoxemia. The aim of the study was to evaluate protective effects of IL-18 and IL-10 in experimentally induced endotoxemia in mice correlating the changes in tissue anti-oxidant enzymes and circulating cytokines. Liver injury was determined by estimation of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), serum nitric oxide (NOx), hepatic anti-oxidant enzyme and cytokine content in LPS (250 microg/kg) induced endotoxemic mice receiving either IL-18 (500 ng/mouse) or IL-10 (600 ng/mouse) treatment. Mice (87% of IL-10 treated and 74% of IL-18 treated) survived when administered prior to LPS challenge. Pre-treatment of mice with either IL-10 or IL-18 followed by LPS, lead to reduction in SGPT and SGOT level, serum NOx, and altered hepatic anti-oxidant enzymes activity and myeloperoxidase activity than the only LPS treated group. Marked reduction in the amounts of LPS-induced hepatic and splenic TNF-u content has been observed after IL-10 pre-treatment. Results suggested that attenuating the induction of TNF-alpha and IFN-gamma and subsequent induction of nitric oxide formation in response to LPS may in part account for efficient protection by IL-18 and IL-10 in the reduction of LPS-induced liver injury.     

Neutralization of IL-18 reduces neutrophil tissue accumulation and protects mice against lethal Escherichia coli and Salmonella typhimurium endotoxemia

In addition to stimulating IFN-gamma synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1beta and the chemokines IL-8 and macrophage inflammatory protein-1alpha. We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice. IL-1beta converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1beta, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium. In contrast, both wild-type and IL-1beta-/- mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-gamma but not IL-1beta in ICE-/- mice is responsible for this resistance. However, IFN-gamma-deficient mice were not resistant to S. typhimurium LPS, suggesting an IFN-gamma-independent role for IL-18. Anti-IL-18 Abs protected mice against a lethal injection of either LPS. Anti-IL-18 treatment also reduced neutrophil accumulation in liver and lungs. The increased survival was accompanied by decreased levels of IFN-gamma and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E. coli LPS, whereas IFN-gamma and TNF concentrations were decreased in treated mice challenged with S. typhimurium. In conclusion, neutralization of IL-18 during lethal endotoxemia protects mice against lethal effects of LPS. This protection is partly mediated through inhibition of IFN-gamma production, but mechanisms involving decreased neutrophil-mediated tissue damage due to the reduction of either chemokines (E. coli LPS) or TNF (S. typhimurium LPS) synthesis by anti-IL-18 treatment may also be involved.     

Increased resistance of LFA-1-deficient mice to lipopolysaccharide-induced shock/liver injury in the presence of TNF-alpha and IL-12 is mediated by IL-10: a novel role for LFA-1 in the regulation of the proinflammatory and anti-inflammatory cytokine balance

Challenge with low doses of LPS together with D-galactosamine causes severe liver injury, resulting in lethal shock (low dose LPS-induced shock). We examined the role of LFA-1 in low dose LPS-induced shock. LFA-1(-/-) mice were more resistant to low dose LPS-induced shock/liver injury than their heterozygous littermates, although serum levels of TNF-alpha and IL-12 were higher in these mice. C57BL/6 mice were not rescued from lethal effects of LPS by depletion of NK1(+) cells, granulocytes, or macrophages, and susceptibility of NKT cell-deficient mice was comparable to that of controls. High numbers of platelets were detected in the liver of LFA-1(+/-) mice after low dose LPS challenge, whereas liver accumulation of platelets was only marginal in LFA-1(-/-) mice. Following low dose LPS challenge, serum levels of IL-10 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, and susceptibility to low dose LPS-induced shock as well as platelet accumulation in the liver of LFA-1(-/-) mice were markedly increased by IL-10 neutralization. Serum levels of IL-10 in LFA-1(+/-) mice were only marginally affected by macrophage depletion. However, in LFA-1(-/-) mice macrophage depletion markedly reduced serum levels of IL-10, and as a corollary, susceptibility of LFA-1(-/-) mice to low dose LPS-induced shock was markedly elevated despite the fact that TNF-alpha levels were also diminished. We conclude that LFA-1 participates in LPS-induced lethal shock/liver injury by regulating IL-10 secretion from macrophages and that IL-10 plays a decisive role in resistance to shock/liver injury. Our data point to a novel role of LFA-1 in control of the proinflammatory/anti-inflammatory cytokine network.     

Contribution of interleukin-12 to the pathogenesis of non-thyroidal illness.

Changes in both central and peripheral thyroid hormone (TH) metabolism occur during illness. These changes, known collectively as non-thyroidal illness, are apparently mediated by the proinflammatory cytokines IL-6, TNFalpha and IFNgamma. IL-12 is involved in regulation of IFNgamma and TNFalpha. The aim of this study was to evaluate the role of IL-12 in TH metabolism during illness. We studied TH metabolism both centrally (in the pituitary) and peripherally (in the liver) in IL-12 knock-out (IL-12 (-/-)) and wild type (WT) mice during illness induced by administration of bacterial endotoxin (LPS). LPS induced a similar decrease in serum T (3), T (4) and liver 5'-DI mRNA expression in IL-12 (-/-) and WT mice with the exception of a smaller reduction of serum T (4) in IL-12 (-/-) mice. In the pituitary, the LPS-induced decline in 5'-DI activity in WT mice was not observed in IL-12 (-/-) mice (p < 0.001), whereas the decrease in DII activity tended to be smaller in IL-12 (-/-) mice (p = 0.066). The lower decrease in pituitary activity of both DI and DII in IL-12 (-/-) mice is possibly related to the lower LPS-induced T (4) decrease. In conclusion, IL-12 is involved in the central regulation of the HPT axis during illness but not in the peripheral regulation.     

Release of soluble ICAM-1 from human lung fibroblasts, aortic smooth muscle cells, dermal microvascular endothelial cells, bronchial epithelial cells, and keratinocytes.

We determined effects of IL-1alpha, TNFalpha and IFNgamma on sICAM-1 release in culture media from human aortic smooth muscle cells (AOSMC), dermal microvascular endothelial cells (DMEC), keratinocytes (KC), bronchial epithelial cells (BEC) and lung fibroblasts (LF) as determined by ELISA. Under basal conditions of cultures for 20 h, low concentrations of sICAM-1 were only detected in the culture media of two (DMEC and BEC) of these cell types. IL-1alpha, TNFalpha and IFNgamma stimulated sICAM-1 from these cells. IFNgamma stimulated more shedding from AOSMC, BEC and KC than IL-1alpha or TNFalpha. TNFalpha enhanced more sICAM-1 release from DEMC than from AOSMC, BEC and LF. IL-1alpha and IFNgamma or TNFalpha and IFNgamma acted synergistically to enhance shedding of sICAM-1 from these cells. The levels sICAM-1 in pathophysiological conditions may influence leukocyte-vascular cell interactions to block leukocyte transmigration to tissue injury sites as a negative feedback mechanism.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

Role for nerve growth factor in the in vivo regulation of glutathione in response to LPS in mice

Since the redox state regulator glutathione (GSH), which influences lipopolysaccharide (LPS) anorexia, may be controlled by cytokines, we studied the roles of tumour necrosis factor-alpha (TNFalpha) and nerve growth factor (NGF) in the GSH response to intraperitoneal (ip) LPS injection in mice. Basal NGF and total reduced GSH (trGSH) levels were up-regulated in brain and liver of TNFalpha-knock-out (KO) mice, and this was associated with attenuated LPS anorexia. The increases in NGF and trGSH presumably contributed to the attenuated anorexia in response to LPS because transgenic mice over-expressing NGF (NGF-tg mice) also had increased trGSH levels and displayed attenuated anorexia compared to the corresponding wild type (WT) mice. Attenuated LPS anorexia in NGF-tg mice was accompanied by reduced serum TNFalpha and IFNgamma levels compared to WT mice. In response to a second injection of LPS, NGF and trGSH levels, but not TNFalpha levels changed. This suggests that in vivo tissue trGSH changes following LPS in LPS-na?ve or LPS-pretreated mice are regulated by NGF rather than TNFalpha. The finding that genetic TNFalpha deficiency did not inhibit the acute trGSH response to LPS supports this interpretation. In sum, the results indicate i) that a decrease or increase in NGF is accompanied by a decrease or increase in trGSH levels and ii) that elevated NGF and/or trGSH levels attenuate some of the responses to LPS such as anorexia and cytokine production.     

Pretreatment with the Gram-positive bacterial cell wall molecule peptidoglycan improves bacterial clearance and decreases inflammation and mortality in mice challenged with Pseudomonas aeruginosa

The objective of this study was to determine if inflammatory tolerance and enhancement of innate immune function could be induced by the Gram-positive cell wall component peptidoglycan (PGN). Male mice (C57BL6/J or C3H/HeJ, 8-12 weeks of age) were given intraperitoneal injections of 1mg PGN on 2 consecutive days. The mice were then challenged with lipopolysaccharide (LPS) or live Pseudomonas aeruginosa (1 x 10(8) colony-forming units) 2 days after the second pretreatment. Mice pretreated with PGN had diminished plasma concentrations of TNFalpha and IFNgamma and elevated concentrations of IL-10 in response to a subsequent LPS or Pseudomonas challenge when compared to untreated controls. Bacterial clearance was improved in mice pretreated with PGN, and mortality in response to a subsequent Pseudomonas challenge was significantly attenuated. PGN pretreatment of LPS-unresponsive mice (C3H/HeJ) verified that the effect of PGN pretreatment was not due to any LPS contamination. We have previously demonstrated that PGN pretreatment induced resistance to a Gram-positive bacterial challenge. The present study extends those results by showing that exposure to the Gram-positive bacterial cell wall component peptidoglycan also induces cross-tolerance to LPS and non-specifically enhances innate immune function in that PGN-pretreated mice had increased resistance to Gram-negative bacterial challenge.     

CpG-induced IFNgamma expands TLR4-specific IL-18 responses in vivo

Serum IL-18 responses to LPS increase after pretreatment with CpG-containing DNA. Compared to saline-pretreated controls, mice pretreated with CpG for two days produced 20-fold more serum IL-18 when challenged with lipopolysaccharide (LPS). In contrast, IFNgamma-deficiency or anti-IFNgamma pretreatment reduced CpG-expanded IL-18 responses to LPS by 67 and 83%, respectively. Mice pretreated with either IFNgamma or CpG comparably increased LPS-inducible serum IL-18 responses. LPS, compared to challenge with other TLR agonists, was best able to trigger high serum IL-18 levels in CpG-pretreated mice and this response was TLR4-dependent. CpG, compared to pretreatment with other TLR agonists, optimally expanded LPS-induced IL-18 responses that correlated with higher levels of circulating IFNgamma levels prior to LPS challenge. High-level serum IL-18 responses were caspase-1-dependent and P2X7 receptor-independent. We conclude that CpG promotes high-level IL-18 synthesis by an IFNgamma-dependent and IFNgamma-sufficient mechanism in vivo that is optimally triggered by LPS.     

Interleukin-1 is not essential for expression of inducible NOS in hepatocytes induced by lipopolysaccharide in vivo

Interleukin (IL)-1 and tumor necrotic factor alpha (TNFalpha) are pivotal in the pathogenesis of endotoxemia. In spite of the in vitro finding that IL-1beta, but not TNFalpha, can induce iNOS mRNA and NO production as a single stimulus in hepatocytes in primary culture, the involvement of IL-1 in iNOS induction in the liver has been less clear in vivo. To address this, we challenged IL-1alpha/beta double-knockout (IL-1alpha/beta(-/-)) and TNFalpha(-/-) mice with lipopolysaccharide (LPS). As compared with wild-type mice, the increases in the plasma NO level measured as nitrite and nitrate and hepatic iNOS were significantly reduced in IL-1alpha/beta(-/-) and TNFalpha(-/-) mice 8 and 12h after the LPS challenge. In the wild-type mice, iNOS protein was first detected in Kupffer cells around the portal vein 2h after LPS challenge; and then it spread to hepatocytes throughout the intralobular region of the liver by 8h. Although the expression of iNOS protein was detected in Kupffer cells of both IL-1alpha/beta(-/-) and TNFalpha(-/-) mice, its level was moderate in hepatocytes of IL-1alpha/beta(-/-) mice, but negligible in those of TNFalpha(-/-) mice, 8h after LPS challenge. Concomitant with the expression of iNOS protein in the liver, Toll-like receptor 4, the signaling receptor for LPS, was expressed in hepatocytes of wild-type and IL-1alpha/beta(-/-) mice, but not of TNFalpha(-/-) mice. These results demonstrate that the expression of Toll-like receptor 4 is well correlated with that of iNOS protein in hepatocytes in vivo after LPS challenge and that IL-1 is not essential for the induction of iNOS in hepatocytes in vivo.     

Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia

Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1β and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.     

Deficient control of Trypanosoma cruzi infection in C57BL/6 mice is related to a delayed specific IgG response and increased macrophage production of pro-inflammatory cytokines

Earlier work in Trypanosoma cruzi-infected C57BL/6 and BALB/c mice revealed an acute disease, of lethal outcome in the former group and lesser severity in BALB/c mice. Fatal course was not accompanied by an increased parasite load, but by a substantial imbalance between pro- and anti-inflammatory cytokine serum levels. To better characterise the mechanisms allowing the host to restrain the infection, we have now studied the specific IgG production and in vitro behaviour of peritoneal macrophages (PMs) when exposed to T. cruzi. BALC/c mice displayed higher serum levels of specific immunoglobulins in the first weeks of acute infection. In vitro infected PMs showed no between-group differences in the number of intracellular parasites, although TNFalpha levels were significantly higher in culture supernatants from C57BL/6 mice. Because an LPS-based pretreatment (desensitisation protocol followed by a sublethal LPS dose) reduced disease severity of C57BL/6 mice, we next explored the features of the in vitro infection in PMs from mice subjected to such protocol. PMs from LPS-pretreated mice had a decreased production of TNFalpha and IL-1beta, becoming more permissive to parasite replication. It is concluded that deficient control of T. cruzi infection in C57BL/6 mice may also involve a less satisfactory specific IgG response and increased TNFalpha production by PMs. Improved disease outcome in LPS-pretreated mice may be associated with the reduced inflammatory cytokine production by PMs, but the impaired ability of these cells to control parasite growth suggests that compensatory mechanisms are operating in the in vivo situation.     

Obesity and IL-6 interact in modulating the response to endotoxemia in mice

Obesity is associated with elevated levels of IL-6. High IL-6 is prognostic of mortality in sepsis, while controversial data link obesity to sepsis outcome. We used Lean and diet-induced obese (DIO) WT and IL-6 KO mice to investigate the interaction between obesity and IL-6 in endotoxemia. Circulating levels of IL-6 were significantly higher in WT DIO versus WT Lean mice receiving LPS (2.5 μg/mouse, ip). Obesity lead to greater weight loss in response to LPS, with IL-6 deficiency being partially protective. Plasma TNFα, IFNγ, Galectin-3 and leptin were significantly elevated in response to LPS and were each differentially affected by obesity and/or IL-6 deficiency. Plasma Galectin-1 and adiponectin were significantly suppressed by LPS, with obesity and IL-6 deficiency modulating the response. However, LPS comparably increased IL-10 levels in each group. Leukopenia with relative neutrophilia and thrombocytopenia developed in each group after injection of LPS, with obesity and genotype affecting the kinetics, but not the magnitude, of the response. Hepatic induction of the acute-phase protein SAA by LPS was not affected by obesity or IL-6 deficiency, although baseline levels were highest in WT DIO mice. Injection of LPS significantly increased hepatic mRNA expression of PAI-1 in Lean WT and Lean KO mice, while it suppressed the high baseline levels observed in the liver of DIO WT and DIO KO mice. Thus, both IL-6 and obesity modulate the response to endotoxemia, suggesting a complex interaction that needs to be considered when evaluating the effect of obesity on the outcome of septic patients.     

Relationships between IFNgamma, IL-6, corticosterone, and Listeria monocytogenes pathogenesis in BALB/c mice

The relationships between Listeria monocytogenes (LM) pathogenesis, based on bacterial load, and serum levels of IL-6, IFNgamma, and corticosterone (CORT) were quantified. Serum IFNgamma levels increased along with the LM burden; however, with LM burdens > or =3 x 10(6) CFU per spleen, the serum IFNgamma level decreased along with a decrease in splenic weight. Serum IL-6 levels exponentially increased with increases of LM, and the CORT level positively correlated with the increase in IL-6 and LM. The serum level of IFNgamma appeared to be a good biomarker of the host's ability to combat the infection only when the LM burden did not exceed a critical level (>3 x 10(6) CFU per spleen). Interestingly, the LM load at which the IFNgamma level began to decline was near the dose at which the IL-6 concentration exponentially increased, suggesting a transition point shift from stress (assessed as CORT level) being immunoenhancing to becoming immunosuppressive. The IL-6:IFNgamma ratio may be a good indicator of disease severity and/or the ability to cope with an infection.     

A synergistic role for IL-1beta and TNFalpha in monocyte-derived IFNgamma inducing activity

Although much is known about classic IFNgamma inducers, little is known about the IFNgamma inducing capability of inflammasome-activated monocytes. In this study, supernatants from LPS/ATP-stimulated human monocytes were analyzed for their ability to induce IFNgamma production by KG-1 cells. Unexpectedly, monocyte-derived IFN inducing activity was detected, but it was completely inhibited by IL-1beta, not IL-18 blockade. Moreover, size-fractionation of the monocyte conditioned media dramatically reduced the IFNgamma inducing activity of IL-1beta, suggesting that IL-1beta requires a cofactor to induce IFNgamma production in KG-1 cells. Because TNFalpha is known to synergize with IL-1beta for various gene products, it was studied as the putative IL-1beta synergizing factor. Although recombinant TNFalpha (rTNFalpha) alone had no IFNgamma inducing activity, neutralization of TNFalpha in the monocyte conditioned media inhibited the IFNgamma inducing activity. Furthermore, rTNFalpha restored the IFNgamma inducing activity of the size-fractionated IL-1beta. Finally, rTNFalpha synergized with rIL-1beta, as well as with rIL-1alpha and rIL-18, for KG-1 IFNgamma release. These studies demonstrate a synergistic role between TNFalpha and IL-1 family members in the induction of IFNgamma production and give caution to interpretations of KG-1 functional assays designed to detect functional IL-18.     

Serum Th1 and Th2 profile cytokine level changes in patients with Graves' ophthalmopathy treated with corticosteroids.

The aim of this study was to estimate the influence of corticosteroids on Th1 and Th2 serum cytokine balance in patients with GO: IFNgamma, TNFalpha, IL-4 and IL-10. Further, we tested the hypothesis of an up-regulation of Th2 immune response during successful treatment with corticosteroids to explain their beneficial effect in Graves' ophthalmopathy. Serum cytokines were detected in three groups of subjects: 20 patients with Graves' disease without ophthalmopathy (Gd), 16 patients with clinical symptoms of ophthalmopathy (GO) (CAS over 3 points, last consultation record for GO less than a year old) and 16 healthy volunteers. Corticosteroid therapy consisted of intravenous infusions of methylprednisolone (MP) (2 series, 3 g each time) and subsequent treatment with oral prednisone (60 mg per day) in a tapering schedule. The serum samples were collected 24 hours before MP, 24 hours after MP, 14 days of treatment with prednisone and at the end of corticosteroid therapy. The levels of IFNgamma, TNFalpha, IL-4 and IL-10 in the serum were determined using ELISA. Statistical significance was estimated by the Mann-Whitney U-test. Our findings show a deviation to systemic Th2 profile cytokines in Graves' disease. In patients with GO, we found a significantly increased serum IL-10 concentration. In corticosteroid-responsive patients, the balance of serum cytokines IL-4/IFNgamma, IL-4/TNFalpha, IL-10/IFNgamma and IL-10/TNFalpha increased and remained upregulated until the end of the study. In non-responders, the balance of serum cytokines studied increased after methylprednisolone but declined markedly during continuation of the therapy with prednisone. In summary, our results show that efficient corticosteroid therapy may be related to its influence on Th2/Th1 profile cytokine balance. The upregulation of serum IL-4 and IL-10 during successful treatment with corticosteroids indicate the possibility of using these cytokines as predictors of the beneficial effect of corticosteroids in Graves' ophthalmopathy.