Markus Haug

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells.

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross‐presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress‐inducible Hsp70 to enhance activation and proliferation of human memory CD4+ T cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen‐specific CD4+ T cells as confirmed by HLA‐DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen‐specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ T cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen‐specific CD4+ T cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ T cells.

Intracellular Mycobacterium avium Intersect Transferrin in the Rab11+ Recycling Endocytic Pathway and Avoid Lipocalin 2 Trafficking to the Lysosomal Pathway

Iron is an essential nutrient for microbes, and many pathogenic bacteria depend on siderophores to obtain iron. The mammalian innate immunity protein lipocalin 2 (Lcn2; also known as neutrophil gelatinase-associated lipocalin, 24p3, or siderocalin) binds the siderophore carboxymycobactin, an essential component of the iron acquisition apparatus of mycobacteria. Here we show that Lcn2 suppressed growth of Mycobacterium avium in culture, and M. avium induced Lcn2 production from mouse macrophages. Lcn2 also had elevated levels and initially limited the growth of M. avium in the blood of infected mice but did not impede growth in tissues and during long-term infections. M. avium is an intracellular pathogen. Subcellular imaging of infected macrophages revealed that Lcn2 trafficked to lysosomes separate from M. avium, whereas transferrin was efficiently transported to the mycobacteria. Thus, mycobacteria seem to reside in the Rab11(+) endocytic recycling pathway, thereby retaining access to nutrition and avoiding endocytosed immunoproteins like Lcn2.

Impaired suppression of synovial fluid CD4+CD25− T cells from patients with juvenile idiopathic arthritis by CD4+CD25+ Treg cells

OBJECTIVE Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25- effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells. METHODS The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in (3) H-thymidine proliferation assays. Cytokine levels were analyzed using Bio-Plex Pro assay. RESULTS Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA-DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells. CONCLUSION Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype.

Lipocalin 2 Imparts Selective Pressure on Bacterial Growth in the Bladder and Is Elevated in Women with Urinary Tract Infection

Competition for iron is a critical component of successful bacterial infections, but the underlying in vivo mechanisms are poorly understood. We have previously demonstrated that lipocalin 2 (LCN2) is an innate immunity protein that binds to bacterial siderophores and starves them for iron, thus representing a novel host defense mechanism to infection. In the present study we show that LCN2 is secreted by the urinary tract mucosa and protects against urinary tract infection (UTI). We found that LCN2 was expressed in the bladder, ureters, and kidneys of mice subject to UTI. LCN2 was protective with higher bacterial numbers retrieved from bladders of Lcn2-deficient mice than from wild-type mice infected with the LCN2-sensitive Escherichia coli strain H9049. Uropathogenic E. coli mutants in siderophore receptors for salmochelin, aerobactin, or yersiniabactin displayed reduced fitness in wild-type mice, but not in mice deficient of LCN2, demonstrating that LCN2 imparts a selective pressure on bacterial growth in the bladder. In a human cohort of women with recurrent E. coli UTIs, urine LCN2 levels were associated with UTI episodes and with levels of bacteriuria. The number of siderophore systems was associated with increasing bacteriuria during cystitis. Our data demonstrate that LCN2 is secreted by the urinary tract mucosa in response to uropathogenic E. coli challenge and acts in innate immune defenses as a colonization barrier that pathogens must overcome to establish infection.

Involvement of CD91 and scavenger receptors in Hsp70-facilitated activation of human antigen-specific CD4+ memory T cells

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress‐inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen‐specific CD4+ T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen‐specific cells were detected combining HLA‐DRB1*0401 or HLA‐DRB1*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen‐specific CD4+ T cell responses.

70-kDa heat shock proteins: Specific interactions with HLA-DR molecules and their peptide fragments

Heat shock protein 70 (HSP70):peptide complexes are involved in MHC class I and class II‐restricted antigen presentation enabling enhanced activation of antigen‐specific T cells. Here, we investigated the potential of bacterial and mammalian HSP70 molecules to interact with peptide fragments from HLA‐DR and the corresponding complete HLA‐DR molecules. Peptide fragments were found to interact with DnaK, the HSP70 homologue from E. coli, but less with stress‐inducible human Hsp70. Only a peptide sequence exclusively found in rheumatoid arthritis‐protective HLA‐DR molecules did not interact with DnaK. Subsequently, we investigated the interaction of complete HLA‐DR molecules with HSP70 and detected a specific HSP70:HLA‐DR interaction, with highest affinity for human stress‐inducible Hsp70. In contrast to the peptide fragments, no allele‐specific differences in Hsp70 affinity were detected with complete HLA‐DR molecules. Interaction with HLA‐DR molecules was increased at lowered pH values, whereas HSP70‐chaperoned peptides were released at acidic pH, thus HSP70 could serve as scanner and carrier for antigenic peptides of self or foreign origin and transfer chaperoned peptides onto MHC class II molecules in acidic late endosomal compartments. Our findings indicate that direct interaction between mammalian HSP70 and HLA‐DR molecules could be involved in the HSP70‐mediated enhancement of MHC class II‐restricted peptide presentation and CD4+ T cell activation.

Possible association of non-binding of HSP70 to HLA-DRB1 peptide sequences and protection from rheumatoid arthritis

Abstract. The β-chains of HLA-DR molecules associated with susceptibility to rheumatoid arthritis (RA) share a common amino acid sequence in their third hypervariable region at position 70–74. This shared epitope could either contribute to preferential binding of a given disease-associated peptide, be involved in disease-induction by molecular mimicry or, by binding to heat shock proteins, influence antigen presentation. It is known that the Escherichia coliMr 70,000 heat shock protein DnaK can bind peptides from the shared epitope. Using a highly sensitive method, we show that peptides covering the third hypervariable region of associated, but also most of the non-associated HLA-DR alleles, bind to DnaK. Similar binding specificities could be found for the constitutively expressed mammalian Mr 70,000 heat shock protein Hsc73 and the inducible mammalian Hsp72. However, peptides containing the amino acid sequence DERAA, found in HLA-DR alleles and strongly associated with protection from RA, did not bind any HSP70. Thus, our results suggest a possible association of non-binding of HSP70 to HLA-DR molecules or its 70–74 fragments and protection from RA.

Dynamics of immune effector mechanisms during infection with Mycobacterium avium in C57BL/6 mice

Opportunistic infections with non‐tuberculous mycobacteria such as Mycobacterium avium are receiving renewed attention because of increased incidence and difficulties in treatment. As for other mycobacterial infections, a still poorly understood collaboration of different immune effector mechanisms is required to confer protective immunity. Here we have characterized the interplay of innate and adaptive immune effector mechanisms contributing to containment in a mouse infection model using virulent M. avium strain 104 in C57BL/6 mice. M. avium caused chronic infection in mice, as shown by sustained organ bacterial load. In the liver, bacteria were contained in granuloma‐like structures that could be defined morphologically by expression of the antibacterial innate effector protein Lipocalin 2 in the adjoining hepatocytes and infiltrating neutrophils, possibly contributing to containment. Circulatory anti‐mycobacterial antibodies steadily increased throughout infection and were primarily of the IgM isotype. Highest levels of interferon‐γ were found in infected liver, spleen and serum of mice approximately 2 weeks post infection and coincided with a halt in organ bacterial growth. In contrast, expression of tumour necrosis factor was surprisingly low in spleen compared with liver. We did not detect interleukin‐17 in infected organs or M. avium‐specific T helper 17 cells, suggesting a minor role for T helper 17 cells in this model. A transient and relative decrease in regulatory T cell numbers was seen in spleens. This detailed characterization of M. avium infection in C57BL/6 mice may provide a basis for future studies aimed at gaining better insight into mechanisms leading to containment of infections with non‐tuberculous mycobacteria.

Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages

Significance Inflammatory signaling is a central mechanism controlling host defenses to pathogens. Members of Mycobacterium avium complex cause disease in immunocompromised patients and in individuals with predisposing lung abnormalities. We provide evidence of a mechanism in human primary macrophages whereby the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) negatively regulates inflammatory responses and thus facilitates intracellular growth of M. avium. Our findings are of high biological and clinical significance, as opportunistic infections with nontuberculous mycobacteria are receiving renewed attention because of increased incidence and difficulties in treatment. Altered Keap1 gene expression may also have vital clinical implications for other inflammation-associated conditions, opening novel research venues for translational research, for instance in the expanding field of host-targeted therapy for infectious diseases. Several mechanisms are involved in controlling intracellular survival of pathogenic mycobacteria in host macrophages, but how these mechanisms are regulated remains poorly understood. We report a role for Kelch-like ECH-associated protein 1 (Keap1), an oxidative stress sensor, in regulating inflammation induced by infection with Mycobacterium avium in human primary macrophages. By using confocal microscopy, we found that Keap1 associated with mycobacterial phagosomes in a time-dependent manner, whereas siRNA-mediated knockdown of Keap1 increased M. avium-induced expression of inflammatory cytokines and type I interferons (IFNs). We show evidence of a mechanism whereby Keap1, as part of an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiquitination and degradation of IκB kinase (IKK)-β thus terminating IKK activity. Keap1 knockdown led to increased nuclear translocation of transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 driving the expression of inflammatory cytokines and IFN-β. Furthermore, knockdown of other members of the Cul3 ubiquitin ligase complex also led to increased cytokine expression, further implicating this ligase complex in the regulation of the IKK family. Finally, increased inflammatory responses in Keap1-silenced cells contributed to decreased intracellular growth of M. avium in primary human macrophages that was reconstituted with inhibitors of IKKβ or TANK-binding kinase 1 (TBK1). Taken together, we propose that Keap1 acts as a negative regulator for the control of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.

Mutations in the substrate binding site of human heat-shock protein 70 indicate specific interaction with HLA-DR outside the peptide binding groove

Heat‐shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I‐ and II‐restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA‐DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA‐DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA‐DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild‐type Hsp70 whereas HLA‐DR binding remains unaffected. The removal of the C‐terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA‐DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA‐DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA‐DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA‐DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70‐chaperoned peptides might thereby be directly transferred into the binding groove of HLA‐DR, so enabling enhanced presentation of the peptide on antigen‐presenting cells and leading to an improved proliferation of responding T cells as shown previously.