Anke Osterloh

Heat shock proteins: linking danger and pathogen recognition

Besides their central function in protein folding and transport within the cell, heat shock proteins (HSP) have been shown to modulate innate and adaptive immune response: (1) HSP mediate uptake and MHC presentation of HSP-associated peptides by antigen-presenting cells (APC). (2) HSP function as endogenous danger signals indicating cell stress and tissue damage to the immune system. (3) HSP bind pathogen-associated molecular pattern (PAMP) molecules and modulate PAMP-induced Toll-like receptor (TLR) signaling. Thus, HSP contribute to both, recognition of “danger” defined as uncontrolled tissue destruction and recognition of dangerous “nonself”. In this review these different aspects of immune stimulation by HSP will be discussed.

Synergistic and Differential Modulation of Immune Responses by Hsp60 and Lipopolysaccharide

Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) γ release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNα production in professional APC. Neutralization of IFNα as well as the absence of functional IFNαβ receptor on APC and T cells interfered with Hsp60-mediated IFNγ secretion in antigen-dependent T cell activation, strongly suggesting that IFNα represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNγ release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.

Toll like receptor 4 mediates cell death in a mouse MPTP model of Parkinson disease

In mammalians, toll-like receptors (TLR) signal-transduction pathways induce the expression of a variety of immune-response genes, including inflammatory cytokines. It is therefore plausible to assume that TLRs are mediators in glial cells triggering the release of cytokines that ultimately kill DA neurons in the substantia nigra in Parkinson disease (PD). Accordingly, recent data indicate that TLR4 is up-regulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in a mouse model of PD. Here, we wished to evaluate the role of TLR4 in the acute mouse MPTP model of PD: TLR4-deficient mice and wild-type littermates control mice were used for the acute administration way of MPTP or a corresponding volume of saline. We demonstrate that TLR4-deficient mice are less vulnerable to MPTP intoxication than wild-type mice and display a decreased number of Iba1+ and MHC II+ activated microglial cells after MPTP application, suggesting that the TLR4 pathway is involved in experimental PD.

Hsp60-mediated T cell stimulation is independent of TLR4 and IL-12

Heat shock protein (Hsp) 60 is thought to function as endogenous danger signal by activating professional antigen-presenting cells (APC) through toll-like receptor (TLR) 4 and CD14, a mechanism that is also used by bacterial LPS. We recently showed that Hsp60 binds LPS and enhances LPS-induced immune stimulation. On the other hand, we also observed immune stimulation by Hsp60 independent of LPS which was partially mediated by Hsp60-induced IFN alpha. Here, we study the mechanisms involved in immune stimulation mediated by endotoxin-free Hsp60. We show that T cell co-stimulation induced by LPS-free Hsp60 was independent of TLR4 and the TLR-associated myeloide differentiation factor 88-signaling pathway. LPS-free Hsp60 did not induce IL-6, IL-12 or tumor necrosis factor alpha production in APC nor were these cytokines needed for Hsp60-mediated T cell co-stimulation in the absence of LPS. In contrast to endotoxin-free Hsp60, T cell co-stimulation induced by LPS or Hsp60/LPS complexes strictly depended on IL-12 and functional TLR-4. Furthermore, we show that LPS-free Hsp60 enhances IFN alpha expression in APC and that this cytokine represents one important mediator in immune stimulation by Hsp60 in the absence of LPS. Taken together, we provide evidence that endotoxin-free Hsp60 and LPS or Hsp60/LPS complexes employ different signaling mechanisms to transduce co-stimulatory signals.

A new view on cutaneous dendritic cell subsets in experimental leishmaniasis

Because of their anatomical distribution epidermal Langerhans cells (LCs) are discussed to be crucial for antigen uptake and subsequent presentation to naïve T cells in skin-draining lymph nodes. The use of LC-specific markers like Langerin or knock-in mice expressing green fluorescent protein under the control of the Langerin promotor now facilitates the dissection of LCs from other dendritic cell (DC) subsets. Surprisingly, current data indicate that LCs are not generally involved in the induction of cellular immune responses. Moreover, the widely accepted paradigm postulating that LCs in principle act as T cell activators is contested by recent publications. Consequently, the biological role of LCs, in particular in cutaneous immune responses, needs to be revisited. The experimental model of leishmaniasis represents a suitable model to study the origin of an antigen-specific T cell response in mice. With this model the transport and presentation of skin derived Leishmania (L.) major antigens can be monitored in vivo. Furthermore, the quality of T cell-DC interactions can be determined. Considering recent progress in LC research we propose a novel concept of LCs in T cell meditated immunity against L. major parasites.

Heat shock protein 60 (HSP60) stimulates neutrophil effector functions

Neutrophil granulocytes belong to the first cells that enter sites of infection, where they eliminate infiltrating pathogens via phagocytosis and the release of antimicrobial mediators. Hence, recruitment of neutrophils and activation of neutrophil microbicidal functions are crucial steps in the early containment of infection. In this study, we show that hHSP60 binds to murine and human PMN strongly and specifically. We demonstrate that HSP60 serves as a chemoattractant and modulates neutrophil functions. Human PMN were incubated with HSP60 alone or prior to stimulation with fMLP or PMA acetate. We observed that HSP60, although not inducing neutrophil release of ROS and degranulation itself, strongly enhanced the production of reactive oxygen induced by PMA and the release of primary granule enzymes induced by both secondary stimuli. This sensitization of PMN was HSP60‐specific. Moreover, PMN that had been preincubated with HSP60 exhibited a marked increase in the uptake of opsonized Escherichia coli in the absence of additional stimuli. Taken together, our results show for the first time that HSP60 modulates antimicrobial effector functions of neutrophil granulocytes. In this way and in agreement with its function as an endogenous danger signal, HSP60, which is released by damaged tissue, may promote early innate defense mechanisms against invading pathogens.

Vaccination with Strongyloides ratti heat shock protein 60 increases susceptibility to challenge infection by induction of Th1 response

The control of strongyloidiasis affecting approximately 100 million people - caused by the gastrointestinal nematode Strongyloides stercoralis - is still based on anti-helminthic treatment. In the current study we analysed the immune response to Strongyloides ratti heat shock protein 60 (srHSP60) as a possible vaccine candidate in the murine system. We show that srHSP60 is a target of both, humoral and cellular response in S. ratti-infected mice. Strikingly, vaccination with srHSP60 without adjuvant or with CFA induced a S. ratti-specific Th1 response in vivo that did not confer protection but slightly increased larval output during challenge infection. Using in vitro T cell stimulation assays we provide further evidence that srHSP60 skewed activated T cells towards a Th1 response that interfered with efficient clearance of S. ratti infection. Vaccination with alum-precipitated srHSP60, in contrast, overruled the Th1-inducing activity intrinsic to srHSP60, induced a Th2 response, and conferred partial protection against a challenge infection. As srHSP60 is actively secreted by S. ratti during all life stages, our findings strongly suggest that srHSP60 induced polarization towards a Th1 response reflects a mechanism of immune evasion by this pathogenic nematode.

CD83 on murine APC does not function as a costimulatory receptor for T cells

The transmembrane glycoprotein CD83 is rapidly upregulated on murine and human DC upon maturation and therefore a costimulatory function for T cell activation has been suggested. Studies employing human APC indeed showed that CD83 expression was positively correlated to the stimulatory capacity of the APC. Murine APC that were CD83 deficient however, did not display a reduced capacity to activate T cells. To elucidate this contradiction, we thoroughly compared the stimulatory capacity of CD83-overexpressing and CD83-deficient APC. Here we show that CD83 expression levels on APC did not affect the capacity of the APC to activate CD8(+) T cells. CD83 expression levels did not significantly affect CD4(+) T cell activation in vivo, but a weak positive correlation of CD83 expression with CD4(+) T cell activation was observed in vitro under suboptimal stimulation conditions. As CD83 expression also positively correlated with MHC-II but not with MHC-I expression, this differential stimulation specifically of CD4(+) T cells could be explained by a higher density of MHC-II peptide complexes on the APC surface. Taken together, our results strongly suggest that CD83 does not deliver crucial costimulatory signals to murine T cells.

Liver Necrosis and Lethal Systemic Inflammation in a Murine Model of Rickettsia typhi Infection: Role of Neutrophils, Macrophages and NK Cells.

Rickettsia (R.) typhi is the causative agent of endemic typhus, an emerging febrile disease that is associated with complications such as pneumonia, encephalitis and liver dysfunction. To elucidate how innate immune mechanisms contribute to defense and pathology we here analyzed R. typhi infection of CB17 SCID mice that are congenic to BALB/c mice but lack adaptive immunity. CB17 SCID mice succumbed to R. typhi infection within 21 days and showed high bacterial load in spleen, brain, lung, and liver. Most evident pathological changes in R. typhi-infected CB17 SCID mice were massive liver necrosis and splenomegaly due to the disproportionate accumulation of neutrophils and macrophages (MΦ). Both neutrophils and MΦ infiltrated the liver and harbored R. typhi. Both cell populations expressed iNOS and produced reactive oxygen species (ROS) and, thus, exhibited an inflammatory and bactericidal phenotype. Surprisingly, depletion of neutrophils completely prevented liver necrosis but neither altered bacterial load nor protected CB17 SCID mice from death. Furthermore, the absence of neutrophils had no impact on the overwhelming systemic inflammatory response in these mice. This response was predominantly driven by activated MΦ and NK cells both of which expressed IFNγ and is considered as the reason of death. Finally, we observed that iNOS expression by MΦ and neutrophils did not correlate with R. typhi uptake in vivo. Moreover, we demonstrate that MΦ hardly respond to R. typhi in vitro. These findings indicate that R. typhi enters MΦ and also neutrophils unrecognized and that activation of these cells is mediated by other mechanisms in the context of tissue damage in vivo.

Persisting Rickettsia typhi Causes Fatal Central Nervous System Inflammation

ABSTRACT Rickettsioses are emerging febrile diseases caused by obligate intracellular bacteria belonging to the family Rickettsiaceae. Rickettsia typhi belongs to the typhus group (TG) of this family and is the causative agent of endemic typhus, a disease that can be fatal. In the present study, we analyzed the course of R. typhi infection in C57BL/6 RAG1−/− mice. Although these mice lack adaptive immunity, they developed only mild and temporary symptoms of disease and survived R. typhi infection for a long period of time. To our surprise, 3 to 4 months after infection, C57BL/6 RAG1−/− mice suddenly developed lethal neurological disorders. Analysis of these mice at the time of death revealed high bacterial loads, predominantly in the brain. This was accompanied by a massive expansion of microglia and by neuronal cell death. Furthermore, high numbers of infiltrating CD11b+ macrophages were detectable in the brain. In contrast to the microglia, these cells harbored R. typhi and showed an inflammatory phenotype, as indicated by inducible nitric oxide synthase (iNOS) expression, which was not observed in the periphery. Having shown that R. typhi persists in immunocompromised mice, we finally asked whether the bacteria are also able to persist in resistant C57BL/6 and BALB/c wild-type mice. Indeed, R. typhi could be recultivated from lung, spleen, and brain tissues from both strains even up to 1 year after infection. This is the first report demonstrating persistence and reappearance of R. typhi, mainly restricted to the central nervous system in immunocompromised mice.