CXCL14 deficiency does not impact the outcome of influenza or Escherichia coli infections in mice.

INTRODUCTION: Chemokines are small proteins that regulate different cellular functions, such as leukocyte activation, chemoattraction and inflammation. The chemokine CXCL14 (BRAK) is a highly conserved gene among species and through evolution. It has been shown that CXCL14 is locally upregulated during viral infections, also, it has been found that this chemokine possesses direct antibacterial activities. Nonetheless, the exact role that CXCL14 plays during infection remains elusive. METHODOLOGY: CXCL14 deficient mice were generated in a C57B6/129 background and followed by phenotypic characterization. Later, the effect of CXCL14 deficiency during influenza infection and E. coli challenge was assessed. RESULTS: Other than a slight weight reduction, CXCL14 deficient mice exhibited no phenotypic alterations. CXCL14 deficiency did not influence the outcome of influenza virus infection or challenge with E. coli, and no statistically significant differences in clinical signs, cellular responses and histopathological findings were observed. CONCLUSIONS: CXCL14 does not seem to play a pivotal role during influenza and E. coli infections of the lung; these results are suggestive of functional overlap between CXCL14 and other chemokines that are present during lung infection.

Expression of a CD200 transgene is necessary for induction but not maintenance of tolerance to cardiac and skin allografts.

CD200, a type 2 transmembrane molecule of the Ig supergene family, can induce immunosuppression in a number of biological systems, as well as promote increased graft acceptance, following binding to its receptors (CD200Rs). Skin and cardiac allograft acceptance are readily induced in transgenic mice overexpressing CD200 under control of a doxycycline-inducible promoter, both of which are associated with increased intragraft expression of mRNAs for a number of genes associated with altered T cell subset differentiation, including GATA-3, type 2 cytokines (IL-4, IL-13), GITR, and Foxp3. Interestingly, some 12-15 days after grafting, induction of transgenic CD200 expression can be stopped (by doxycycline withdrawal), without obvious significant effect on graft survival. However, neutralization of all CD200 expression (including endogenous CD200 expression) by anti-CD200 mAb caused graft loss, as did introduction of an acute inflammatory stimulus (LPS, 10 microg/mouse, delivered by i.p. injection). We conclude that even with apparently stably accepted tissue allografts, disruption of the immunoregulatory balance by an intense inflammatory stimulus can cause graft loss.

A comparison of the biological properties of small molecular weight agonists and antagonists of CD200:CD200R interactions.

Our laboratory and others have documented in some detail the immunological consequences which follow from interaction of the ubiquitously expressed molecule CD200 with its receptor(s) CD200R (expressed predominantly on cells of myeloid and lymphoid origin). In particular, there is evidence that these interactions lead to immunosuppressive signals which modulate graft rejection responses; decrease the manifestations of arthritis in rodent models; diminish mast cell mediator release in models of allergic disease; and favour the growth of tumors in both mice and humans. The development of small molecular weight agonists (and/or antagonists) of these interactions would thus likely have significant clinical importance. The data reported herein characterizes several such molecules in a number of rodent models.

An interaction between CD200 and monoclonal antibody agonists to CD200R2 in development of dendritic cells that preferentially induce populations of CD4+CD25+ T regulatory cells.

In previous studies we reported that while interaction between the relatively ubiquitously expressed molecule CD200 and one of its receptors, CD200R1, resulted in direct suppression of alloreactivity, engagement of alternate receptors led instead to altered differentiation of dendritic cells (DCs) from marrow precursors, which could in turn foster development of Foxp3(+) regulatory T cells. We have explored this effect of engagement of alternate receptors by using a monoclonal agonist Ab to CD200R2 and investigating expression of TLRs on DCs induced in vivo and in vitro after CD200 stimulation in mice in which the gene encoding CD200R1 was deleted. CD200 stimulation was achieved by using either a soluble form of CD200 (CD200Fc) or overexpression of CD200 as a doxycycline-inducible transgene. Although broadly similar effects were seen, consistent with the hypothesis that triggering of CD200R2 does produce DCs with a characteristic TLR repertoire, there are subtle differences in suppression of alloreactivity achieved by CD200 delivered in these two manners, which is consistent with a complexity of CD200:CD200R engagement not previously appreciated.

Peptides of CD200 modulate LPS-induced TNF-alpha induction and mortality in vivo.

Interaction of the ubiquitously expressed molecule CD200 with its receptor(s) CD200R, expressed on cells of myeloid and lymphoid origin, delivers immunoregulatory signals that modulate inflammation in a number of diseases, including transplant rejection and arthritis. A number of isoforms of CD200R have been described in mice with distinct function. We have synthesized small (9-13 mer) peptides defining distinct regions of CD200, and asked whether different peptides would function as agonists and/or antagonists of different CD200R isoforms. The assays used to characterize these functions include a model of inflammation and tumor necrosis factor-alpha production induced following lipopolysaccharide administration in vivo, and mixed leukocyte cultures in vitro. Discrete agonist and antagonist peptides were defined for different CD200Rs, which suggests this approach may have some clinical utility.

Mice lacking CD200R1 show absence of suppression of lipopolysaccharide-induced tumor necrosis factor-alpha and mixed leukocyte culture responses by CD200.

BACKGROUND: CD200:CD200R interactions deliver immunoregulatory signals. A family of CD200Rs (CD200R1-5) has been described, and engagement of CD200R1 by its ligand CD200 suppresses LPS-induced macrophage cytokine production, decreases alloimmune responses in vivo and in vitro, and suppresses collagen-induced arthritis. METHODS: We generated C57BL/6 mice lacking the genomic exons encoding the extracellular domains of the CD200R1 molecule using transformation of ES cells and explored cell subtypes and immune responses in these mice. RESULTS: Myeloid cells/splenocytes from CD200R1(-/-) mice were not stained in FACS by anti-CD200R1 mAb. Stimulation of splenic tumor necrosis factor-alpha production by lipopolysaccharide was enhanced relative to control (+/+) mice and was not suppressed by addition of exogenous CD200Fc. Modulation of alloreactivity in mixed leukocyte cultures by CD200Fc depended upon CD200R1+ stimulatory cells, although maximal immunoregulation by CD200Fc occurred only when CD200R1+ T responder cells also were used. CD200Fc failed to suppress graft rejection in CD200R1(-/-) mice. CONCLUSION: CD200:CD200R1 plays an immunoregulatory role in vivo.

Molecular identification of vaginal lactobacilli isolated from Bulgarian women.

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in comparison with a set of 21 reference strains based on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of them to be classified as belonging to one of the following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.

Augmented Induction of CD4+CD25+ Treg using monoclonal antibodies to CD200R.

BACKGROUND: CD200 is a transmembrane protein delivering immunoregulatory signals after engagement of CD200R. A family of CD200Rs exist (CD200R1-4) with different tissue expression and functional activity. In the presence of anti-CD200R2/3 monoclonal antibodies (mAbs), bone-marrow cells cultured in the presence of (interleukin [IL]-4+granulocyte-macrophage colony-stimulating factor) differentiate into dendritic cells (DCs), which induce CD4+CD25+ Treg. The effect of these mAbs on Treg induced in anti-CD3 activated thymocyte cultures is unknown. METHODS: BL/6 bone-marrow cells were cultured with GMC-SF and IL-4 in the presence/absence of anti-CD200Rs to generate DCs that induced Treg in C3H lymph-node T cells. Treg were also induced in anti-CD3/CD28-activated C3H thymocytes. Treg activity was assayed by (1) suppression of mixed leukocyte culture (MLC) in cultures using C3H stimulator spleen cells and BL/6 stimulator cells, and (2) by expression of the transcription factor, Foxp3. RESULTS: Addition of anti-CD200R2/3 mAbs (but not anti-CD200R1) to bone-marrow cultures led to generation of DCs that induced a CD4+CD25+ (Treg) population inhibiting MLCs (C3H cells stimulated with C57BL/6 cells) in a cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and transforming growth factor (TGF)-beta-dependent manner. Anti-CD200R2, but not anti-R1/R3, augmented induction of Foxp3-expressing Treg from anti-CD3/CD28 activated thymocytes. Suppression in MLCs by anti-CD200R1 mAbs was dependent on IL-10 and TGF-beta. CONCLUSIONS: Unlike anti-CD200R1, anti-CD200R2 both promotes development of DCs with capacity to induce Treg and directly augments thymocyte production of Treg.

Augmented induction of CD4+CD25+ Treg using monoclonal antibodies to CD200R.

BACKGROUND: CD200 is a transmembrane protein delivering immunoregulatory signals after engagement of CD200R. A family of CD200Rs exist (CD200R1-4) with different tissue expression and functional activity. In the presence of anti-CD200R2/3 monoclonal antibodies (mAbs), bone-marrow cells cultured in the presence of (interleukin [IL]-4+granulocyte-macrophage colony-stimulating factor) differentiate into dendritic cells (DCs), which induce CD4CD25Treg. The effect of these mAbs on Treg induced in anti-CD3 activated thymocyte cultures is unknown. METHODS: BL/6 bone-marrow cells were cultured with GMC-SF and IL-4 in the presence/absence of anti-CD200Rs to generate DCs that induced Treg in C3H lymph-node T cells. Treg were also induced in anti-CD3/CD28-activated C3H thymocytes. Treg activity was assayed by (1) suppression of mixed leukocyte culture (MLC) in cultures using C3H stimulator spleen cells and BL/6 stimulator cells, and (2) by expression of the transcription factor, Foxp3. RESULTS: Addition of anti-CD200R2/3 mAbs (but not anti-CD200R1) to bone-marrow cultures led to generation of DCs that induced a CD4CD25 (Treg) population inhibiting MLCs (C3H cells stimulated with C57BL/6 cells) in a cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and transforming growth factor (TGF)-beta-dependent manner. Anti-CD200R2, but not anti-R1/R3, augmented induction of Foxp3-expressing Treg from anti-CD3/CD28 activated thymocytes. Suppression in MLCs by anti-CD200R1 mAbs was dependent on IL-10 and TGF-beta. CONCLUSIONS: Unlike anti-CD200R1, anti-CD200R2 both promotes development of DCs with capacity to induce Treg and directly augments thymocyte production of Treg.