Ulrich Steinhoff

TLR-Activated B Cells Suppress T Cell-Mediated Autoimmunity

TLR sense microbial infections, and control activation of immune responses. Dendritic cells, macrophages, and B lymphocytes express TLR and the TLR-signaling adaptor protein MyD88. The impact of TLR-activated B cells on T cell-mediated inflammation is unknown. In this study, we have used mice carrying B cell-restricted deficiencies in MyD88 or in distinct TLR to examine the impact of TLR-activated B cells on a T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE). We demonstrate that TLR-signaling in B cells suppresses inflammatory T cell responses (both Th1 and Th17), and stimulates recovery from EAE. Only certain TLR are required on B cells for resolution of EAE, and these are dispensable for disease initiation, indicating that a category of TLR agonists preferentially triggers a suppressive function in B cells and thereby limits autoimmune disease. The TLR agonists controlling the regulatory function of B cells are provided by components of Mycobacterium tuberculosis present in the adjuvant. Thus, MyD88 signaling in B cells antagonizes MyD88 signaling in other cells, which drives differentiation of Th17 cells and is required for induction of EAE. Altogether, our data indicate that B cells link recognition of microbial products via TLR to suppression of a T cell-mediated autoimmune disease.

Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis

Mycobacterium bovis bacille Calmette–Guérin (BCG) is the most widely used live bacterial vaccine. However, limited information is available correlating route and dose of vaccination and induction of specific T cell responses with protection against tuberculosis. We compared efficacy of oral and systemic vaccination and correlated vaccine-induced T cell responses with protection in experimental tuberculosis of mice. After oral and systemic vaccination, we observed profound differences in persistence and dissemination of BCG and frequencies and location of specific IFN-γ-secreting CD4+ and CD8+ T cells. Yet, both vaccination routes caused comparable levels of protection against aerosol challenge with Mycobacterium tuberculosis. Protection correlated best with rapid accumulation of specific CD8+ T cells in infected tissues of challenged mice. In contrast, specific IFN-γ production by CD4+ T cells reflected the load of M. tuberculosis rather than the strength of protection. Our data question the measurement of IFN-γ secretion by CD4+ T cells and emphasize the need for new biomarkers for evaluation of tuberculosis vaccine efficacies.

MyD88/TLR9 mediated immunopathology and gut microbiota dynamics in a novel murine model of intestinal graft-versus-host disease

Background The bacterial microflora aggravates graft-versus-host-disease (GvHD) after allogeneic stem cell transplantation, but the underlying mechanisms of manifestations of intestinal GvHD (iGvHD) in the gut remain poorly understood. Aim To analyse the gut flora composition and the impact of bacterial sensing via Toll-like receptors (TLRs) in iGvHD. Methods By mimicking clinical low-intensity conditioning regimens used in humans, a novel irradiation independent, treosulfan and cyclophosphamide-based murine allogeneic transplantation model was established. A global survey of the intestinal microflora by cultural and molecular methods was performed, the intestinal immunopathology in TLR-deficient recipient mice with iGvHD investigated and finally, the impact of anti-TLR9 treatment on iGvHD development assessed. Results The inflammatory responses in iGvHD were accompanied by gut flora shifts towards enterobacteria, enterococci and Bacteroides/Prevotella spp. Analysis of iGvHD in MyD88-/-, TRIF-/-, TLR2/4-/-, and TLR9-/- recipient mice showed that bacterial sensing via TLRs was essential for iGvHD development. Acute iGvHD was characterised by increasing numbers of apoptotic cells, proliferating cells, T cells and neutrophils within the colon. These responses were significantly reduced in MyD88-/-, TLR2/4-/-, TRIF-/- and TLR9-/- mice, as compared with wild-type controls. However, TRIF-/- and TLR2/4-/- mice were not protected from mortality, whereas TLR9-/- mice displayed increased survival rates. The important role of TLR9-mediated immunopathology was independently confirmed by significantly reduced macroscopic disease symptoms and colonic apoptosis as well as by reduced T-cell and neutrophil numbers within the colon after treatment with a synthetic inhibitory oligonucleotide. Conclusions These results emphasise the critical role of gut microbiota, innate immunity and TLR9 in iGvHD and highlight anti-TLR9 strategies as novel therapeutic options.

Proteasome-mediated degradation of IκBα and processing of p105 in Crohn disease and ulcerative colitis

Enhanced NF-kappaB activity is involved in the pathology of both forms of inflammatory bowel disease (IBD), Crohn disease (CD) and ulcerative colitis (UC). Here we analyzed the mechanism of proteasome-mediated NF-kappaB activation in CD and UC. Our studies demonstrate that the subunit composition and the proteolytic function of proteasomes differ between UC and CD. High expression of the immunoproteasome subunits beta1i and beta2i is characteristic of the inflamed mucosa of CD. In line with this, we found enhanced processing of NF-kappaB precursor p105 and degradation of inhibitor of NF-kappaB, IkappaBalpha, by immunoproteasomes isolated from the mucosa of CD patients. In comparison with healthy controls and CD patients, UC patients exhibited an intermediate phenotype regarding the proteasome-mediated processing/degradation of NF-kappaB components. Finally, increased expression of the NF-kappaB family member c-Rel in the inflamed mucosa of CD patients suggests that p50/c-Rel is important for IFN-gamma-mediated induction of immunoproteasomes via IL-12-driven Th1 responses. These findings suggest that distinct proteasome subunits influence the intensity of NF-kappaB-mediated inflammation in IBD patients.

Rapid Neutrophil Response Controls Fast-Replicating Intracellular Bacteria but Not Slow-Replicating Mycobacterium tuberculosis

Being one of the first cells to invade the site of infection, neutrophils play an important role in the control of various bacterial and viral infections. In the present work, the contribution of neutrophils to the control of infection with different intracellular bacteria was investigated. Mice were treated with the neutrophil-depleting monoclonal antibody RB6-8C5, and the time course of infection in treated and untreated mice was compared by using intracellular bacterial species and strains varying in virulence and replication rate. The results indicate that neutrophils are crucial for the control of fast-replicating intracellular bacteria, whereas early neutrophil effector mechanisms are dispensable for the control of the slow-replicating Mycobacterium tuberculosis.

Autoimmune Intestinal Pathology Induced by hsp60-Specific CD8 T Cells

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.

Heat‐Shock Protein 60: Implications for Pathogenesis of and Protection against Bacterial Infections

In this review we have focused on antigenic features of hsp 60 related to: its ubiquitous distribution in the biosphere; its extraordinary homology among various bacteria; its high conservation from prokaryotic to eukaryotic cells; and its abundant expression under stress situations occurring during infection. These unique features make hsp 60 an excellent candidate antigen relevant to protection and pathogenesis of bacterial infections and, perhaps in a broader sense, to surveillance and autoimmunity. We will briefly discuss these possibilities in the following. Acquired resistance. If we assume that bacterial organisms contain some thousand different proteins which all represent potential antigens, the frequency of T cells with specificity for mycobacterial hsp 60 appears surprisingly high. Although, during the course of infection, high levels of hsp may be induced in bacteria, mere abundance appears to be an important though insufficient explanation. In addition, constant boosting by similar hsp 60 cognates from various microbes with which humans come into contact may contribute to dominance. This could easily explain the occurrence of hsp 60-specific T cells in healthy individuals with no clinical history of mycobacterial infections. Involvement of more sophisticated mechanisms, such as the affinity of hsp to other proteins, cannot be excluded (Flynn et al. 1989). Yet dominance does not necessarily mean protection and definite proof that hsp are protective antigens is lacking. Perhaps the immune response against epitopes shared by various mycobacterial pathogens represents a first line of defence preceding a more specific immune response. Such broadly reactive antigens would not qualify as prime candidates for vaccine design. Immunesurveillance. T cells with specificity for epitopes shared by bacterial and human hsp 60 are readily demonstrable and stressed host cells are recognized by hsp 60-specific T cells. Such T lymphocytes are endowed with the capacity to identify host cells stressed by a variety of assaults such as inflammation, infection, trauma, or transformation. Although it has been claimed that hsp-reactive gamma/delta T cells are particularly destined for such surveillance functions (Born et al. 1990, Asarnow et al. 1988), alpha/beta T cells could also participate. Pathogenesis. The mechanisms causing pathogenesis should be similar to those underlying protection and surveillance. In the former case bacterial hsp would be responsible for both induction of immunity and expression of pathogenic reactions; in the latter case an immune response stimulated by conserved regions of bacterial hsp 60 would be converted against a host-derived cognate.(ABSTRACT TRUNCATED AT 400 WORDS)

Targeting the proteasome: partial inhibition of the proteasome by bortezomib or deletion of the immunosubunit LMP7 attenuates experimental colitis

Background and aims Inflammatory bowel disease (IBD), comprising Crohn´s disease and ulcerative colitis, is characterised by chronic relapsing inflammation of the gut. Increased proteasome activity, associated with the expression of immunoproteasomes, was found to enhance proinflammatory signalling and thus promotes inflammation in patients with IBD. The aim of this study was to explore whether modulation of the proteasomal activity is a suitable therapeutic approach to limit inflammation in colitis. Methods This concept was assessed in two different experimental set-ups. Development of dextran sodium sulfate (DSS)-induced colitis was tested (1) in lmp7−/− mice lacking the immunoproteasome subunit LMP7 and (2) in wild-type (WT) mice treated with the proteasome inhibitor bortezomib. Results Compared with WT mice, lmp7−/− mice develop significantly attenuated colitis due to reduced nuclear factor-κB (NF-κB) signalling in the absence of LMP7. Further, treatment with bortezomib revealed dose-dependent amelioration of DSS-induced inflammation. In both approaches modulation of the proteasome activity limited the secretion of proinflammatory cytokines and chemokines. Consequently, infiltration of the colon by neutrophils and expansion of inflammatory T helper 1 (Th1) and Th17 T cells was diminished and thus prevented excessive tissue damage. Conclusions It was demonstrated that modulation of the proteasome activity is effective in attenuating experimental colitis. The results reveal that reduction of the proteasome activity either by partial inhibition with bortezomib or by specifically targeting the immunoproteasome subunit LMP7 is a suitable treatment of intestinal inflammation.

Link between Organ-specific Antigen Processing by 20S Proteasomes and CD8+ T Cell–mediated Autoimmunity

Adoptive transfer of cross-reactive HSP60-specific CD8+ T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8+ T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of α and β chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8+ T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses.

c-Rel is crucial for the induction of Foxp3(+) regulatory CD4(+) T cells but not T(H)17 cells

The NF‐κB/Rel family member c‐Rel was described to be required for the development of TH1 responses. However, the role of c‐Rel in the differentiation of TH17 and regulatory CD4+Foxp3+ T cells (Treg) remains obscure. Here, we show that in the absence of c‐Rel, in vitro differentiation of pro‐inflammatory TH17 cells is normal. In contrast, generation of inducible Treg (iTreg) within c‐Rel‐deficient CD4+ T cells was severely hampered and correlated to reduced numbers of Foxp3+ T cells in vivo. Mechanistically, in vitro conversion of naive CD4+ T cells into iTreg was crucially dependent on c‐Rel‐mediated synthesis of endogenous IL‐2. The addition of exogenous IL‐2 was sufficient to rescue the development of c‐Rel‐deficient iTreg. Thus, c‐Rel is essential for the development of Foxp3+ Treg but not for TH17 cells via regulating the production of IL‐2.