Heiner von Buttlar

CD4(+) FoxP3(+) regulatory T cells suppress fatal T helper 2 cell immunity during pulmonary fungal infection.

The opportunistic fungal pathogen Cryptococcus neoformans causes lung inflammation and fatal meningitis in immunocompromised patients. Regulatory T (Treg) cells play an important role in controlling immunity and homeostasis. However, their functional role during fungal infection is largely unknown. In this study, we investigated the role of Treg cells during experimental murine pulmonary C. neoformans infection. We show that the number of CD4+FoxP3+ Treg cells in the lung increases significantly within the first 4 weeks after intranasal infection of BALB/c wild‐type mice. To define the function of Treg cells we used DEREG mice allowing selective depletion of CD4+FoxP3+ Treg cells by application of diphtheria toxin. In Treg cell‐depleted mice, stronger pulmonary allergic inflammation with enhanced mucus production and pronounced eosinophilia, increased IgE production, and elevated fungal lung burden were found. This was accompanied by higher frequencies of GATA‐3+ T helper (Th) 2 cells with elevated capacity to produce interleukin (IL)‐4, IL‐5, and IL‐13. In contrast, only a mild increase in the Th1‐associated immune response unrelated to the fungal infection was observed. In conclusion, the data demonstrate that during fungal infection pulmonary Treg cells are induced and preferentially suppress Th2 cells thereby mediating enhanced fungal control.

Conventional Bone Marrow-Derived Dendritic Cells Contribute to Toll-Like Receptor-Independent Production of Alpha/Beta Interferon in Response to Inactivated Parapoxvirus Ovis

ABSTRACT Parapoxvirus ovis (PPVO) is a member of the Poxviridae family and belongs to the genus Parapoxvirus. It displays only limited homology with orthopoxviruses and has some molecular features such as an unusual high GC content distinct from orthopoxviruses. Inactivated PPVO (iPPVO) displays strong immunostimulatory capacities mediating antiviral activity in vivo. The role of dendritic cells (DC) and the pattern recognition receptors and signaling requirements responsible for immunostimulation by iPPVO are unknown. We demonstrate here that bone marrow-derived plasmacytoid DC (BM-pDC) and bone marrow-derived conventional DC (BM-cDC) secrete alpha/beta interferon (IFN-α/β) in response to iPPVO. Furthermore, iPPVO induces tumor necrosis factor alpha (TNF-α) and interleukin-12/23p40 (IL-12/23p40) release and major histocompatibility complex class II (MHC-II), MHC-I, and CD86 upregulation by bone marrow-derived DC (BMDC). After engulfment, iPPVO is located in endosomal compartments and in the cytosol of BMDC. iPPVO elicits IFN-α/β by Toll-like receptor (TLR)-independent pathways in BM-cDC, since IFN-α/β release does not require myeloid differentiation primary response gene 88 (MyD88) or TIR-domain containing adaptor protein inducing interferon (TRIF). In contrast, iPPVO-induced TNF-α release and enhanced expression of MHC-I and CD86 but not of MHC-II by BMDC chiefly requires MyD88 but not TLR2 or TLR4. Induction of IFN-α by iPPVO in BM-cDC occurred in the absence of IFN regulatory factor 3 (IRF3) but required the presence of IRF7, whereas iPPVO-triggered IFN-β production required the presence of either IRF7 or IRF3. These results provide the first evidence that iPPVO mediates its immunostimulatory properties by TLR-independent and TLR-dependent pathways and demonstrate an important role of cDC for IFN-α/β production.

Canine CD4+CD8+ double positive T cells in peripheral blood have features of activated T cells

In dogs a CD4(+)CD8(+) double positive T cell subpopulation exists that has not been phenotypically defined yet. We demonstrate that canine CD4(+)CD8(+) T cells are mature CD1a(-) and TCRαβ(+) T cells. To analyse the activation potential of CD4(+)CD8(+) T cells, PBMC from dogs vaccinated against canine distemper virus (CDV) were re-stimulated with CDV. Upon antigen-specific stimulation, the CD4(+)CD8(+) T cell fraction increases and consists nearly exclusively of proliferated cells. Similarly, other features of activated effector/memory T cells such as up-regulation of CD25 and MHC-II as well as down-regulation of CD62L (L-selectin) were observed in CD4(+)CD8(+) T cells after stimulation. Canine CD4(+)CD8(+) T cells are less abundant, but more heterogeneous than porcine ones, comprising a small proportion expressing the β chain of CD8 in addition to the CD8α chain, like human CD4(+)CD8(+) T cells. In summary, this analysis provides the basis for functional characterisation of the in vivo relevance of CD4(+)CD8(+) T cells in T-cell mediated immunity.

Comparative and retrospective molecular analysis of Parapoxvirus (PPV) isolates.

Species members of the genus Parapoxvirus (PPV) within the family Poxviridae cause contagious pustular dermatitis in small ruminants (Orf virus, ORFV) and mostly mild localized inflammation in cattle (bovine papular stomatitis virus, BPSV and pseudocowpox virus, PCPV). All PPVs are known to be zoonotic, leading to circumscribed skin lesions in humans, historically known as milkers nodules. Human PPV isolates are often ill defined concerning their allocation to an animal origin. Here we present a comparative molecular analysis of a unique collection of 21 historic and recent human and animal PPV cell culture isolates (and two PPV DNA samples). Cell culture PPV propagation was restricted to primary ruminant fibroblasts and was strictly kept at low passages to avoid genomic changes by in vitro influences. For molecular arrangement of the isolate DNAs and their attribution to established PPV species DNA fragments of the PPVs were generated by two different discriminating PCR protocols, targeting the major part of the open reading frame (ORF) 011 (B2L gene) and the complete ORF 032. Multiple sequence alignments and phylogenetic analysis of both genes resulted in affiliation to the known PPV species. The sequences from the ORF 032 allowed discrimination of the isolate DNAs at a higher resolution. Human PPV isolates could be clearly assigned to the PPV species belonging to the reported or assumed animal host of transmission. For the first time, a whole PPV genome sequence comparison of a human biopsy derived virus (B029) and its ovine counterpart (B015) originating from a defined Orf outbreak in Germany is provided, revealing their well conserved relationship. Thus human PPVs can be molecularly retraced to the PPV species indicating the animal of transmission. After transmission to the human host, molecular conservation of the animals virus peculiarities indicative for a PPV species became evident.

Absence of in vitro innate immunomodulation by insect-derived short proline-rich antimicrobial peptides points to direct antibacterial action in vivo

Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well‐known immunomodulatory activities of murine cathelicidin‐related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro‐inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS‐mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections. Copyright

Canine CD4+CD8+ double-positive T cells can develop from CD4+ and CD8+ T cells

For a long time the expression of the CD4 and CD8 receptor on peripheral blood T cells was thought to be mutually exclusive. However, in canine peripheral blood, similar to other species as swine or human for example, mature CD4(+)CD8(+) double-positive (dp) T cells exist which simultaneously express both surface receptors and have features of activated T cells. Canine CD4(+)CD8(+)dp T cells are heterogeneous and can be divided into three subpopulations by their intensity of CD4 and CD8α expression: CD4(bright)CD8α(bright), CD4(dim)CD8α(bright) and CD4(dim)CD8α(dim). The number of CD4(+)CD8α(+)dp T cells increases after in vitro stimulation of canine peripheral blood mononuclear cells (PBMC) raising the question of their progenitor(s). Thus, the aim of our study was to characterize the progenitor(s) of canine CD4(+)CD8α(+)dp T cells. By cell tracing experiments we identified both CD4(+) single-positive (sp) and also CD8α(+)sp T cells as progenitors of canine CD4(+)CD8α(+)dp T cells after in vitro stimulation. CD4(+)sp T cells almost exclusively upregulate a CD8αα homodimer, whereas CD8α(+)sp T cells can become CD4(+)CD8αβ(+) or CD4(+)CD8αα(+). Even in the absence of other cells, highly purified CD4(+)sp T cells can become double-positive upon in vitro stimulation, whereas highly purified CD8α(+)sp T cells fail to do so. However, CD8α(+)sp T cells can additionally express CD4 when stimulated in the presence of CD4(-)CD8α(-) double-negative (dn) cells or more efficiently when stimulated in the presence of CD4(+)sp T cells. Soluble factors secreted by CD4(+)sp T cells are sufficient for the upregulation of CD4 on CD8α(+)sp T cells, but direct cell-cell contact between CD4(+)sp and CD8α(+)sp T cells is more efficient. mRNA analysis shows that additional CD4 expression on CD8α(+)sp T cells results from de novo synthesis. Thus, uptake of soluble CD4 or trogocytosis is less likely as mechanism for generation of canine double-positive T cells. CD4(+)CD8α(+)dp T cells are highly activated independent of their origin except when generated in coculture of CD8α(+)sp T cells with CD4(-)CD8α(-)dn cells. Overall, in dog, CD4(+)sp T cells are the more potent progenitors of CD4(+)CD8α(+)dp T cells compared to CD8α(+)sp T cells.

Analysis of asthma patients for cryptococcal seroreactivity in an urban German area

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised patients but is also able to asymptomatically infect immunocompetent individuals. C. neoformans is found ubiquitously especially in urban areas where it is spread by pigeons, and fungal exposure may predispose for asthma development already at an early age, as soon as confronted with pigeon droppings. In the study presented here, we investigated the presence of specific immunoglobulin G (IgG) against C. neoformans in sera from patients suffering from asthma in comparison to a healthy control cohort, accrued from the Leipzig Research Centre for Civilization Diseases (LIFE). For serological analysis we developed a flow cytometry (FACS) based assay specific for an acapsular strain of C. neoformans to comprehensively analyze different cryptococcal serotypes. Compared with the non-asthmatic cohort, asthmatics exhibited, as expected, an elevated level of total serum immunoglobulin E (IgE), whereas the IgG seroreactivity against C. neoformans was not significantly different among the two groups (P = .118). Nevertheless, there was a trend toward increased Cryptococcus-specific IgG antibodies in the serum of asthmatics. Additionally, in male asthmatics an increased IgG-mediated seroreactivity compared to female asthmatics was found. This points to a higher prevalence of subclinical C. neoformans infection in male asthmatics and may support the hypothesis of C. neoformans as a risk factor for the development of asthma in urban areas.

Identification of Toll-like receptor 9 as parapoxvirus ovis-sensing receptor in plasmacytoid dendritic cells.

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and co-stimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.

Canine peripheral blood CD4+CD8+ double-positive T cell subpopulations exhibit distinct T cell phenotypes and effector functions

Canine peripheral blood CD4+CD8α+ double-positive (dp) Tcells represent a small population of T lymphocytes that are not only derived from single-positive (sp) CD4+ but also from CD8+ Tcells. Dp Tcells can be subdivided in three different subpopulations according to their expression levels of CD4 and CD8α, i.e. the CD4dimCD8αbright, CD4brightCD8αbright, and the CD4brightCD8αdim subsets. Previously we could show that total canine peripheral CD4+CD8α+ dp Tcells have features of activated Tcells. Here we demonstrate that the individual dp Tcell subsets distinctly differ in their activation status and phenotype. Thus, among the dp Tcell subsets the CD4dimCD8αbright subpopulation has the lowest and the CD4brightCD8αbright subset the highest frequency of CD25+ cells pointing to the CD4brightCD8αbright subset with the highest activation status. Consistent with that, analysis of CD44 vs. CD62L expression demonstrates that the CD4brightCD8αbright subset contains the highest frequencies of effector-memory Tcells (TEM). Upon in vitro stimulation, the CD4brightCD8αbright and CD4dimCD8αbright dp Tcell subsets show the highest frequencies of IFN-γ producing cells. Expression of granzyme B was found exclusively in the CD4dimCD8αbright dp Tcell subset indicating cytotoxic potential of this dp Tcell subset. Furthermore, T-bet expression was restricted to the CD4dimCD8αbright subset, while Foxp3+ regulatory Tcells were detected in the CD4brightCD8αdim dp Tcell subset. In conclusion, canine dp Tcells are phenotypically and functionally heterogeneous consistent with the diverse characteristics of their single-positive CD4+ and CD8+ Tcell progenitors. The data provide the basis for future in vivo studies to elucidate the role of individual dp Tcell subsets in host defense and/or immune pathological diseases of dogs.