Thomas W. Jungi

TOLL-like receptors linking innate and adaptive immune response.

Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive immunity, which is mediated by B and T lymphocytes, recognises pathogens by rearranged high affinity receptors. However, the establishment of adaptive immunity is often not rapid enough to eradicate microorganisms as it involves cell proliferation, gene activation and protein synthesis. More rapid defense mechanisms are provided by innate immunity, which recognises invading pathogens by germ-line-encoded pattern recognition receptors (PRR). Recent evidence shows that this recognition can mainly be attributed to the family of TOLL-like receptors (TLR). Binding of pathogen-associated molecular patterns (PAMP) to TLR induces the production of reactive oxygen and nitrogen intermediates (ROI and RNI), pro-inflammatory cytokines, and up-regulates expression of co-stimulatory molecules, subsequently initiating the adaptive immunity. In this review, we will summarize the discovery and the critical roles of the TLR family in host defense, briefly allude to signaling mechanisms mediating the response to TLR ligands, and will provide an update on current knowledge regarding the ligand specificity of these receptors and their role in immunity of domestic animals, particularly cattle.

Female barn owls (Tyto alba) advertise good genes.

The good genes hypothesis of sexual selection postulates that ornamentation signals superior genetic quality to potential mates. Support for this hypothesis comes from studies on male ornamentation only, while it remains to be shown that female ornamentation may signal genetic quality as well. Female barn owls (Tyto alba) display more black spots on their plumage than males. The expression of this plumage trait has a genetic basis and it has been suggested that males prefer to mate with females displaying more black spots. Given the role of parasites in the evolution of sexually selected traits and of the immune system in parasite resistance, we hypothesize that the extent of female plumage ‘spottiness’ reflects immunological defence. We assessed the genetic variation in specific antibody production against a non–pathogenic antigen among cross–fostered nestlings and studied its covariation with the plumage spottiness of genetic parents. The magnitude of the antibody response was positively correlated with the plumage spottiness of the genetic mother but not of the genetic father. Our study thereby provides the first experimental support, to our knowledge, for the hypothesis that female ornamentation signals genetic quality.

Differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with Toll-like receptor agonists

Toll‐like receptors (TLR) have been described as partially sharing signalling pathways but showing unique ligand specificity and tissue distribution. Here, the response of bovine macrophages (Mφ) and dendritic cells (DC), both derived from monocytes, was compared by exposing them to the TLR‐specific ligands lipopolysaccharide, poly(I:C)‐double‐stranded RNA, and CpG‐DNA, as well as inactivated Gram‐negative and Gram‐positive bacteria, shown to bind to TLR. The production of NO, superoxide anion, interleukin‐10 (IL‐10), IL‐12 and tumour necrosis factor (TNF) was determined. Compared to monocytes, Mφ expressed more TLR2 and similar levels of TLR4 mRNA transcripts, as analysed by quantitative polymerase chain reaction, whereas DC expressed reduced amounts. Although both DC and Mφ recognized the TLR ligands, dramatic differences were seen in their reaction pattern to them. Both cell types responded with the production of TNF, but DC produced more IL‐12, whereas Mφ produced more IL‐10, regardless of the TLR agonist used. Co‐stimulation with interferon‐γ influenced the amount of cytokine production, but did not alter the cell type‐specific response pattern. Compared to Mφ, DC produced > 10 times less NO upon triggering with TLR ligands. In addition, DC produced superoxide anion to opsonized and non‐opsonized zymosan, but not to phorbol 12‐myristate 13‐acetate, a response pattern confirmed for human Mφ and DC, respectively. Different protein kinase C isoforms and extracellular signal‐regulated kinase patterns were detected in cell lysates of resting and stimulated Mφ and DC. Collectively, our results point to profound differences in pathogen‐derived signal–response coupling occurring commensurate with distinct functions carried out by Mφ or DC.

Exploration of stress-induced immunosuppression in chickens reveals both stress-resistant and stress-susceptible antigen responses

In the present study, depriving chickens of foraging material was shown to induce stress. The impact of this type of stress on the immune response was compared with feeding of corticosterone (1.5 mg per bird per day), a hormone known to be immunosuppressive and to be the major stress hormone of chickens. Corticosterone feeding induced stress as revealed by higher heterophil/lymphocyte (H/L) ratios, longer tonic immobility (TI) reaction, reduced body weight gain and reduced egg production. Blood corticosterone levels were increased. Corticosterone feeding decreased the antibody response to tetanus toxoid and SRBC, DTH to PPD from Mycobacterium tuberculosis and the inflammatory response to PHA. Housing chickens on slats also induced chronic stress, as evidenced by increased H/L ratios, prolonged TI duration and decreased egg production. Corticosterone levels were slightly but not significantly enhanced. This novel form of chronic stress strongly suppressed humoral and cellular immune responses as evidenced by lower antibody titers to sheep red blood cells (SRBC) and tetanus toxoid (TT) decreased DTH reaction to PPD and inflammatory reaction to PHA in the skin. In contrast, the antibody response to human serum albumin (HSA) was neither influenced by corticosterone feeding nor by keeping the birds on slats. Even the combination of corticosterone feeding and housing the birds on slats did not significantly impair antibody responses to HSA. In conclusion, the present study showed that chronic stress induced by depriving the birds of foraging material led to a similar impairment of humoral and cell-mediated immunity as did feeding with corticosterone. More importantly, it showed for the first time that depending on the antigen tested, there are stress-resistant and stress-susceptible antigen responses.

BVDV and innate immunity

Infection with bovine viral diarrhea virus (BVDV) is prevalent in the cattle population worldwide. The virus exists in two biotypes, cytopathic and non-cytopathic, depending on the effect of the viruses on cultured cells. BVDV may cause transient and persistent infections which differ fundamentally in the hosts antiviral immune response. Transient infection may be due to both cytopathic and non-cytopathic biotypes of BVDV and leads to a specific immune response. In contrast, only non-cytopathic BVD viruses can establish persistent infection as a result of infection of the embryo early in its development. Persistent infection is characterized by immunotolerance specific for the infecting viral strain. In this paper we discuss the role of innate immune responses in the two types of infection. In general, both transient and persistent infections are associated with an increased frequency of secondary infections. Associated with the increased risk of such infections are, among others, impaired bacteria killing and decreased chemotaxis. Interestingly, non-cytopathic BVDV fails to induce interferon type I in cultured bovine macrophages whereas cytopathic biotypes readily trigger this response. Cells infected with non-cytopathic BVDV are also resistant to induction of interferon by double stranded RNA, a potent interferon inducer signalling the presence of viral replication in the cell. Thus, non-cytopathic BVDV may dispose of a mechanism suppressing a key element of the antiviral defence of the innate immune system. Since interferon is also important in the activation of the adaptive immune response, suppression of this signal may be essential for the establishment of persistent infection and immunotolerance.

Possible mechanisms of intravenous immunoglobulin treatment in childhood idiopathic thrombocytopenic purpura (ITP).

Childhood ITP is an acquired condition which often represents a sequela of infection. It is characterized by marked thrombocytopenia despite an increased platelet production in the bone marrow, resulting from a drastically shortened lifespan of platelets. In most patients an increased amount of platelet-associated IgG is observed. Whereas the disease normally regresses regardless of therapeutic intervention, it lasts for more than one year in about 10% of the cases and becomes a condition comparable to chronic ITP observed in adults. Although the topic of ITP has been reviewed extensively [15, 17, 22], etiology and pathogenesis of ITP remain obscure. The observation of a rapid increase of platelet numbers in 13 children with r iP after high intravenous dose of ImmunoglobulinSRK (IgG-SRK, identical with Sandoglobulin) [14], prorated us to discuss immunological aspects of childhood ITP and possible mechanisms by which high doses of intravenous immunoglobulin mayfavourably influence this condition.

poly(I:C) and LPS induce distinct IRF3 and NF‐κB signaling during type‐I IFN and TNF responses in human macrophages

Macrophages play major roles in the onset of immune responses and inflammation by inducing a variety of cytokines such as TNF and IFN‐β. The pathogen‐associated molecular pattern, polyinosinic‐polycytidylic acid [poly(I:C)], and LPS were used to study type‐I IFN and TNF responses in human macrophages. Additionally, activation of the key signaling pathways, IFN‐regulatory factor 3 (IRF3) and NF‐κB, were studied. We found that TNF production occurred rapidly after LPS stimulation. LPS induced a strong IFN‐β mRNA response within a short time‐frame, which subsided at 8 h. The IFN‐stimulated genes (ISGs), ISG56 and IFN‐inducible protein 10, were strongly induced by LPS. These responses were associated with NF‐κB and IRF3 activation, as shown by IRF3 dimerization and by nuclear translocation assays. poly(I:C), on the other hand, induced a strong and long‐lasting (>12 h) IFN‐β mRNA and protein response, particularly when transfected, whereas only a protracted TNF response was observed when poly(I:C) was transfected. However, these responses were induced in the absence of detectable IRF3 and NF‐κB signaling. Thus, in human macrophages, poly(I:C) treatment induces a distinct cytokine response when compared with murine macrophages. Additionally, a robust IFN‐β response can be induced in the absence of detectable IRF3 activation.

Upregulation of toll-like receptors in chronic enteropathies in dogs

BACKGROUND Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown. HYPOTHESIS That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. ANIMALS Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. METHODS Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs. RESULTS There were significant differences (P< or = .017) in expression of TLR2, 4, and 9 between the 6 sampled locations in healthy control dogs (lymph node > small intestine > or = colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores. CONCLUSIONS AND CLINICAL IMPORTANCE Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.

Toll-like receptor-4 is involved in eliciting an LPS-induced oxidative burst in neutrophils.

The lipopolysaccharide (LPS) receptor complex of mononuclear phagocytes is composed of Toll-like receptor-4 (TLR4), MD-2 and CD14. Other phagocyte populations may express similar LPS receptors. The transmembrane glycoprotein TLR4 was shown to induce or upregulate a variety of gene products, which collectively are the mediators of an LPS effect. In this study, an involvement of TLR4 in mediation of an oxidative burst was determined using murine peritoneal exsudate neutrophils and lucigenin-enhanced chemiluminescence (CL). The CL response was dependent on the LPS dose and the presence of serum, putatively a source of lipopolysaccharide-binding protein (LBP). In the absence of serum, a CL signal was elicited by 4 microg/ml LPS in peritoneal exsudate cells (PEC) from TLR4-sufficient (C3H/HeN) but not TLR4 deficient (C3H/HeJ) mice. The signal obtained in PEC from TLR4-sufficient mice was completely abrogated by superoxide dismutase (SOD), which indicated that the response depended on the formation of superoxide anion, and was also seen in purified neutrophils but not purified macrophages (Mphi). In the presence of serum, lower LPS concentrations (e.g. 40 ng/ml) elicited a strong CL response in PEC from TLR4-sufficient, and a weak signal in cells from TLR-4-deficient mice. This suggests that TLR4 engagement is involved in promoting an oxidative burst in murine neutrophils.

INDUCIBLE NITRIC OXIDE SYNTHASE OF MACROPHAGES. PRESENT KNOWLEDGE AND EVIDENCE FOR SPECIES-SPECIFIC REGULATION

An important mechanism by which macrophages (M phi) halt the growth of and eliminate a broad array of intracellular pathogens is the production of nitric oxide (NO). NO generation is catalyzed by inducible nitric oxide synthase (iNOS) converting arginine into citrulline and NO. In murine M phi, iNOS activity is regulated largely at the transcriptional level. LPS and IFN-gamma induce iNOS, IL-4 and TGF-beta down-regulate LPS or IFN-gamma induced iNOS. In human M phi, iNOS cannot be induced by conventional activating regimes in vitro. We studied iNOS induction in ruminant monocytes and M phi from various sources (bone marrow, alveolar lavage, peripheral blood) and found that there is a species-specific and differentiation stage-dependent pattern of iNOS regulation in vitro. Notably, cattle M phi and monocytes respond to distinct signals by iNOS expression. Goat monocytes and M phi resemble human, pig and rabbit M phi in that upon treatment with conventional activating stimuli, they express less iNOS than unstimulated murine or bovine M phi and fail to generate detectable amounts of nitrite and nitrate.