Cytohesin Binder and Regulator (Cybr) is Not Essential for T- and Dendritic-Cell Activation and Differentiation

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.

Langerin(+) Dermal Dendritic Cells Are Critical for CD8(+) T Cell Activation and IgH gamma-1 Class Switching in Response to Gene Gun Vaccines

The C-type lectin langerin/CD207 was originally discovered as a specific marker for epidermal Langerhans cells (LC). Recently, additional and distinct subsets of langerin(+) dendritic cells (DC) have been identified in lymph nodes and peripheral tissues of mice. Although the role of LC for immune activation or modulation is now being discussed controversially, other langerin(+) DC appear crucial for protective immunity in a growing set of infection and vaccination models. In knock-in mice that express the human diphtheria toxin receptor under control of the langerin promoter, injection of diphtheria toxin ablates LC for several weeks whereas other langerin(+) DC subsets are replenished within just a few days. Thus, by careful timing of diphtheria toxin injections selective states of deficiency in either LC only or all langerin(+) cells can be established. Taking advantage of this system, we found that, unlike selective LC deficiency, ablation of all langerin(+) DC abrogated the activation of IFN-gamma producing and cytolytic CD8(+) T cells after gene gun vaccination. Moreover, we identified migratory langerin(+) dermal DC as the subset that directly activated CD8(+) T cells in lymph nodes. Langerin(+) DC were also critical for IgG1 but not IgG2a Ab induction, suggesting differential polarization of CD4(+) T helper cells by langerin(+) or langerin-negative DC, respectively. In contrast, protein vaccines administered with various adjuvants induced IgG1 independently of langerin(+) DC. Taken together, these findings reflect a highly specialized division of labor between different DC subsets both with respect to Ag encounter as well as downstream processes of immune activation. The Journal of Immunology, 2011, 186: 1377-1383.

Structure activity studies of mast cell activation and hypotension induced by neuropeptide Y (NPY), centrally truncated and C-terminal NPY analogues

1 Neuropeptide-induced histamine release is thought to occur via receptor-independent mechanisms, with net charge and lipophilicity being important factors.

Influence of alpha-helicity, amphipathicity and D-amino acid incorporation on the peptide-induced mast cell activation

Mast cell activation by polycationic substances is believed to result from a direct activation of G protein alpha subunits and it was suggested that the adaption of amphipathic, alpha-helical conformations would allow the peptide to reach the cytosolic compartment to interact with G proteins (Mousli et al., 1994, Immunopharmacology 27, 1, for review). We investigated the histamine-releasing activity of model peptides as well as analogues of magainin 2 amide and neuropeptide Y with different amphipathicities and alpha-helix content on rat peritoneal mast cells. Amphipathic helicity is not a prerequisite for mast cell activation. Moreover, non-helical magainin peptides with high histamine-releasing activity were less active in the liberation of carboxyfluoresceine from negatively charged liposomes, indicating that peptide-induced mast cell activation and peptide-induced membrane perturbation do not correlate. In contrast to the negligible influence of the secondary structure, amino acid configuration may exert a striking influence on peptide-induced mast cell activation. Thus histamine-release by substance P was markedly impaired when the L-amino acids in the positively charged N-terminal region were replaced by D-amino acids, with [D-Arg(1)]substance P being the most inactive substance P diastereoisomer.

EFFECT OF IN-VIVO AND IN-VITRO LOVASTATIN TREATMENT ON MAST-CELL ACTIVATION

The hydroxymethylglutaryl coenzmye A (HMG CoA) reductase inhibitor lovastatin is used to treat hyperlipidaemia. This agent prevents the isoprenylation of some proteins involved in signal transduction processes and inhibits IgE-receptor-linked mediator release from RBL-2H3 cells. In this study the effect of in vivo and in vitro administration of lovastatin on histamine release from rat peritoneal mast cells was examined. Lovastatin (4 mg/kg/day for 2 weeks) inhibited histamine release induced by concanavalin A (con A) from rat peritoneal mast cells of Hooded-Lister rats and both homozygous lean and obese Zucker rats. In contrast, release induced by antirat IgE (anti-IgE) was only significantly inhibited in cells derived from Hooded-Lister rats and that induced by compound 48/ 80 was not altered. Lovastatin (20 mu M, 24 h, in vitro) caused a significant inhibition of the subsequent histamine release to con A, anti-IgE and compound 48/80 but not to the calcium ionophore A 23187. It is important to determine whether such inhibitory effects are also observed after the chronic, clinical administration of lovastatin and other HMG CoA reductase inhibitors.

Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation

Strains of the Burkholderia cepacia complex have emerged as a serious threat to patients with cystic fibrosis due to their ability to infect the lung and cause, in some patients, a necrotizing pneumonia that is often lethal. It has recently been shown that several strains of the B. cepacia complex can escape intracellular killing by free-living amoebae following phagocytosis. In this work, the ability of two B. cepacia complex strains to resist killing by macrophages was explored. Using fluorescence microscopy, electron microscopy and a modified version of the gentamicin-protection assay, we demonstrate that B. cepacia CEP021 (genomovar VI), and Burkholderia vietnamiensis (previously B. cepacia genomovar V) CEP040 can survive in PU5-1.8 murine macrophages for a period of at least 5 d without significant bacterial replication. Furthermore, bacterial entry into macrophages stimulated production of tumour necrosis factor and primed them to release toxic oxygen radicals following treatment with phorbol myristoyl acetate. These effects were probably caused by bacterial LPS, as they were blocked by polymyxin B. Infected macrophages primed with interferon gamma produced less nitric oxide than interferon-gamma-primed uninfected cells. We propose that the ability of B. cepacia to resist intracellular killing by phagocytic cells may play a role in the pathogenesis of cystic fibrosis lung infection. Our data are consistent with a model where repeated cycles of phagocytosis and cellular activation without bacterial killing may promote a deleterious inflammatory response causing tissue destruction and decay of lung function.

A role of cellular prion protein in programming T-cell cytokine responses in disease.

The cellular prion protein (PrPC) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrPC in adaptive immunity has been a particular conundrum: increased expression of cell surface PrPC has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8–24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrPC is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP0/0 T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF- and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrPC is a potentially important molecule influencing T-cell activation and effector function.

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Defects in acute responses to TLR4 in Btk-deficient mice result in impaired dendritic cell-induced IFN-γ production by natural killer cells

This study defines a critical role for Btk in regulating TLR4-induced crosstalk between antigen presenting cells (APCs) and natural killer (NK) cells. Reduced levels of IL-12, IL-18 and IFN-? were observed in Btk-deficient mice and ex vivo generated macrophages and dendritic cells (DCs) following acute LPS administration, whilst enhanced IL-10 production was observed. In addition, upregulation of activation markers and antigen presentation molecules on APCs was also impaired in the absence of Btk. APCs, by virtue of their ability to produce IL-12 and IL-18, are strong inducers of NK-derived IFN-?. Co-culture experiments demonstrate that Btk-deficient DCs were unable to drive wild-type or Btk-deficient NK cells to induce IFN-? production, whereas these responses could be restored by exogenous administration of IL-12 and IL-18. Thus Btk is a critical regulator of APC-induced NK cell activation by virtue of its ability to regulate IL-12 and IL-18 production in response to acute LPS administration.

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.

Innate immune activation in neovascular Age-related Macular Degeneration

Purpose: To compare white blood cell populations from persons with neovascular age-related macular degeneration (nAMD) with that of age-matched controls.

Methods: Immunophenotyping for white blood cell populations (including CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocytes, CD4 and CD8 T-lymphocytes, CD56 natural killer cells, CD19 B-lymphocytes and CD16+HLA-DR- neutrophils), chemokine receptor expression analysis (CX3CR1 and CCR2) as well as cell activation analysis (MHC-II, HLA-DR, CD62L, STAT3) was performed using samples of peripheral blood from nAMD patients and age- and gender-matched controls.

Results: The percentage of CD4+ T cells was significantly reduced while the percentage of CD11b+ cells and CD16+HLA-DR- neutrophils was significantly increased in nAMD patients compared to controls. The percentage of classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes was similar between nAMD patients and controls, however there was a significant increase of CX3CR1 on the intermediate monocyte subset and on CD16+HLA-DR- neutrophils in nAMD compared to controls. HLA-DR was significantly increased in all monocyte subsets in nAMD compared to controls. Activation of Signal Transducer and Activator of Transcription 3 (STAT3) was significantly increased in nAMD patients compared to controls following stimulation with IL6.

Conclusions: Our results suggest an increased activation of the innate immune system in patients with nAMD. A better understanding of the role of the innate immune system in the pathogenesis of nAMD may help identify novel biomarkers and thus development of improved therapeutic strategies.