Chronic pneumonia despite adaptive immune response to Mycobacterium bovis BCG in MyD88-deficient mice

To assess the role of Toll-like receptor (TLR) signalling in host response to mycobacterial infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with the vaccine strain Mycobacterium bovis (BCG), and the immune response and bacterial burden were investigated. Macrophages and dendritic cells from MyD88-deficient mice stimulated in vitro with BCG mycobacterial antigens produced very low levels of proinflammatory cytokines, while the expression of costimulatory molecules such as CD40 and CD86 was preserved. Upon systemic infection with BCG (2 x 10(6) CFU i.v.) MyD88-deficient mice developed confluent chronic pneumonia with two log higher CFU than wild-type mice. Interestingly, the infection was controlled in liver and spleen and there was efficient systemic T-cell priming with high IFNgamma production by CD4+ splenic T cells in MyD88-deficient mice. Lung infiltrating cells showed IFNgamma production by pulmonary CD4+ T cells upon specific restimulation, and a reduced capacity to produce nitric oxide and IL-10. In summary, despite the dramatic reduction of the innate immune response, MyD88-deficient mice were able to mount an efficient T-cell response to mycobacterial antigens, which was however insufficient to control infection in the lung, resulting in chronic pneumonia in MyD88-deficient mice.     

Protein kinase C-theta is required for development of experimental cerebral malaria

Cerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ-deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ-deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8(+) T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ-deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8(+) T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM.     

The novel immunosuppressant FTY720 induces peripheral lymphodepletion of both T- and B-cells in cynomolgus monkeys when given alone, with Cyclosporine Neoral or with RAD

OBJECTIVE: FTY720 is a new immunosuppressant active in transplantation models, which modulates lymphocyte recirculation, leading to transient peripheral lymphopenia and increased lymphocytes in lymph nodes and Peyer's patches. Here, we investigated the susceptibility of cynomolgus monkeys to FTY720 given orally either alone or in combination with two other immunosuppressants, Cyclosporin Neoral or RAD, as an introductory study to transplantation protocols. METHODS: Each of the three phases of the study comprised a 3-week treatment period with FTY720 administered daily orally at 0.3, 0.1 or 0.03 mg/kg/day, respectively, followed by a 3-week recovery. FTY720 was given as single compound during the first week and in combination with Neoral at 20 mg/kg/day p.o. or RAD at 0.5 mg/kg/day p.o. during the subsequent 2 weeks. MAIN FINDINGS: These treatment regimen were well tolerated, except for some body weight loss at high FTY720 dose (0.3 mg/kg/day). FTY720 treatment resulted in a rapid decrease of white blood cell counts which reached a plateau after 3 days. A decrease in both T- and B-lymphocyte counts by up to 80-90% was seen with FTY720 doses of 0.1 and 0.3 mg/kg/day. FTY720 blood levels, both trough levels and AUC(0-24 h), showed a linear relationship with FTY720 dose. The reduction in lymphocyte counts was not directly proportional to FTY720 blood levels. The exposure to FTY720 significantly increased upon coadministration of Neoral. This pharmacokinetic interaction was not observed for coadministration of RAD. However, the peripheral lymphodepletion was slightly increased after coadministration of RAD but not of Neoral. This may be related to the intrinsic effects of RAD on hematopoietic cells. CONCLUSIONS: FTY720 given orally was effective in terms of peripheral T- and B-lymphodepletion and was well tolerated in cynomolgus monkeys even in combination with Cyclosporine Neoral or RAD, indicating that such combination protocols could be used in allo- and xenotransplantation in this species. However, the data indicate a potentiation of FTY720 exposure by CsA coadministration and additional lymphodepletion by coadministration of FTY720 and RAD which should be carefully monitored.     

Endogenous leukemia inhibitory factor attenuates endotoxin response

Leukemia inhibitory factor (LIF) is induced in inflammation and likely plays a regulatory role. Using LIF-deficient mice (LIF-/-), we report here that endogenous LIF has a protective role in endotoxic shock and host defence. LIF-/- mice have heightened sensitivity to LPS in a LPS/D-galactosamine (D-Gal) sensitization model compared to wild-type mice (LIF+/+), enhanced thrombocytopenia and leukopenia, with increased hepatic necrosis, neutrophil sequestration in the lung and accelerated mortality. These findings correlated with 10-fold higher tumour necrosis factor-alpha (TNFalpha) and interleukin-6 (IL-6) serum levels and reduced IL-10 production in LIF-/- mice in response to LPS. Therefore, endogenous LIF attenuates the endotoxic shock response, enhances the expression of basal acute phase proteins and IL-10 production, which downregulates TNFalpha synthesis and release and thereby confers partial protection to endotoxemia.     

Monoclonal antibody technology for cyclosporine monitoring

Monitoring blood levels of Cyclosporine (CsA) has been the basis for adjusting individual dosage regimens in the clinic. Radioimmunoassays using polyclonal antisera reacted with CsA and some CsA metabolites, leading to overestimation when compared with high-performance liquid chromatographic measurements of CsA. Monoclonal antibodies (mAbs) have the potential to discriminate between closely related molecules. MAbs with high affinity for CsA have been prepared and their fine-specificity characterized by cross-reactivity studies using a large series of CsA-derivatives. According to the known sites of metabolism on the CsA molecule and to its three-dimensional structure, it was possible to predict which mAb would be suitable for recognizing native Cs specifically.     

Elucidation of toll-like receptor and adapter protein signaling in vascular dysfunction induced by gram-positive Staphylococcus aureus or gram-negative Escherichia coli

Pathogens contain specific pathogen-associated molecular patterns, which activate pattern recognition receptors of the innate immune system such as Toll-like receptors (TLRs). Although there is a clear evidence of how macrophages sense pathogens, we know less about such processes in vessels. This is critical to understand because activation of vascular cells and the subsequent induction of inflammatory genes by bacteria are crucial events in the development of septic shock. In the current study we have used genetically modified mice to investigate the role of TLRs, adapter proteins, tumor necrosis factor alpha (TNFalpha), and nitric oxide synthase II (NOSII) in vascular dysfunction induced by Gram-positive (Staphylococcus aureus) or Gram-negative (Escherichia coli) bacteria. Our data show that Gram-positive S. aureus or Gram-negative E. coli causes vascular dysfunction via the induction of NOSII. For S. aureus, this process requires TLR2, TLR6, myeloid differentiation factor 88 (MyD88) adapter-like, MyD88, and TNF, but not TLR4 or TLR1. Vascular dysfunction induced by E. coli requires TLR4 but has no requirement for TLR2, TLR1, TLR6, or TNF, and a partial but incomplete requirement of MyD88 and TIR domain-containing adapter inducing interferon-beta. Staphylococcus aureus induced NOSII protein expression in vascular smooth muscle cells but not in macrophages, whereas E. coli induced NOSII in both cell types. Our data are the first to establish the definitive roles of specific TLRs in the sensing of Gram-positive and Gram-negative bacteria by vessels and demonstrate that macrophages and blood vessels may differ in their response to pathogens.     

IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice

The molecular mechanisms of acute lung injury resulting in inflammation and fibrosis are not well established. Here we investigate the roles of the IL-1 receptor 1 (IL-1R1) and the common adaptor for Toll/IL-1R signal transduction, MyD88, in this process using a murine model of acute pulmonary injury. Bleomycin insult results in expression of neutrophil and lymphocyte chemotactic factors, chronic inflammation, remodeling, and fibrosis. We demonstrate that these end points were attenuated in the lungs of IL-1R1– and MyD88-deficient mice. Further, in bone marrow chimera experiments, bleomycin-induced inflammation required primarily MyD88 signaling from radioresistant resident cells. Exogenous rIL-1β recapitulated a high degree of bleomycin-induced lung pathology, and specific blockade of IL-1R1 by IL-1 receptor antagonist dramatically reduced bleomycin-induced inflammation. Finally, we found that lung IL-1β production and inflammation in response to bleomycin required ASC, an inflammasome adaptor molecule. In conclusion, bleomycin-induced lung pathology required the inflammasome and IL-1R1/MyD88 signaling, and IL-1 represented a critical effector of pathology and therapeutic target of chronic lung inflammation and fibrosis.     

Role of lipopolysaccharide in the induction of type I interferon-dependent cross-priming and IL-10 production in mice by meningococcal outer membrane vesicles

We investigated the contribution of lipopolysaccharide (LPS) to adjuvant properties of native outer membrane vesicles (NOMV), a vaccine candidate for meningococcal B disease. NOMV induce the maturation of and cytokine production by murine bone marrow-derived dendritic cells through both toll-like receptors (TLR) 2 and 4 which are mostly dependent on the signalling adaptor MyD88. NOMV are also able to induce B cell proliferation in splenocytes from LPS-hyporesponsive mice. However, induction of IL-10 and type I interferon-dependent, antigen-specific and IFN_γ-secreting CD8⁺ cytotoxic T lymphocyte responses in vivo by NOMV requires LPS. The importance of LPS in the induction of IL-10 and functional cross-priming has implications for NOMV-based vaccine and adjuvant development.