Thorsten Joeris

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.

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.

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.

Immunoproteasomes Are Essential for Clearance of Listeria monocytogenes in Nonlymphoid Tissues but Not for Induction of Bacteria-Specific CD8+ T Cells

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8+ T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8+ T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8+ T cell epitopes. I-proteasome-deficient mice (β5i−/− mice) exhibited normal frequencies of L. monocytogenes-specific CD8+ T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8+ T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.

Lymph-borne CD8α + dendritic cells are uniquely able to cross-prime CD8 + T cells with antigen acquired from intestinal epithelial cells

Cross-presentation of cellular antigens is crucial for priming CD8+ T cells, and generating immunity to intracellular pathogens—particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103+ CD11b− CD8α+ DCs cross-present IEC-derived ovalbumin to CD8+ OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC–ovalbumin was limited to the CD11c+ MHCIIhi CD8α+ migratory DCs, but absent from all other subsets, including the resident CD8αhi DCs. Crucially, delivery of purified CD8α+ LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8+ T cells in vivo. Finally, in 232-4 mice treated with R848, CD8α+ LDCs were uniquely able to cross-prime interferon γ-producing CD8+ T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α+ intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8+ T cells. They may therefore represent an important target for the development of antiviral vaccinations.

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αβ(+) and CD4(+)CD8αα(+) T cells; the latter requiring β8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

Comparative Expression Analysis and Characterization of 20S Proteasomes in Human Intestinal Tissues: The Proteasome Pattern as Diagnostic Tool for IBD Patients

Background: The diagnostic differentiation between Crohns disease (CD) and ulcerative colitis (UC) is sometimes difficult. To date, there are no serological markers that are specific and sensitive enough to differentiate between these 2 diseases. Early and safe prediction of the inflammatory bowel disease (IBD) type is of great importance for the specific treatment of IBD patients. We thus analyzed and compared the expression of catalytic proteasome subunits in the gut of mice and in the normal and inflamed intestines of CD and UC patients and assessed whether the subunit pattern is suitable for diagnostic differentiation. Methods: The 20S proteasomes were isolated from surgical tissue specimens derived from terminal ileum and colon of IBD patients and controls. Spots of 20S proteasomes separated by 2D electrophoresis were analyzed by mass spectrometry. Quick detection of catalytic β2, β2i, and β5i subunits was performed by incubating proteasomes with a biotinylated inhibitor (AdaK(Bio)Ahx3L3VS) and subsequently by streptavidin‐horseradish peroxide. Results: 20S proteasomes were isolated from the human liver, colon, and terminal ileum. Low expression of the immunosubunits β1i and β2i was found in the liver and colon but high amounts in the small intestine. In colon and liver β5i was found to be associated with the constitutive β1, β2 subunits, indicating the existence of mixed proteasomes. Further, inflammation in CD but not UC patients induced massive upregulation of β1i and β2i in the colon and terminal ileum, indicating the importance of this protein complex as a disease marker. Conclusions: We here show that CD and UC patients display a characteristic pattern of proteasome subunit composition which can be used as diagnostic tool to differentiate between CD and UC. (Inflamm Bowel Dis 2008)

Diversity and functions of intestinal mononuclear phagocytes

The intestinal lamina propria (LP) contains a diverse array of mononuclear phagocyte (MNP) subsets, including conventional dendritic cells (cDC), monocytes and tissue-resident macrophages (mφ) that collectively play an essential role in mucosal homeostasis, infection and inflammation. In the current review we discuss the function of intestinal cDC and monocyte-derived MNP, highlighting how these subsets play several non-redundant roles in the regulation of intestinal immune responses. While much remains to be learnt, recent findings also underline how the various populations of MNP adapt to deal with the challenges specific to their environment. Understanding these processes should help target individual subsets for ‘fine tuning’ immunological responses within the intestine, a process that may be of relevance both for the treatment of inflammatory bowel disease (IBD) and for optimized vaccine design.

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors.

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or ‘double’ tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the ‘double’ tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.

c‐Rel promotes type 1 and type 17 immune responses during Leishmania major infection

Recent studies demonstrated the crucial role of c‐Rel in directing Treg lineage commitment and its involvement in T helper 1 (Th1) cell‐mediated autoimmune inflammation. We thus wondered whether these opposite functions of c‐Rel influence the course of antiparasitic immune responses against Leishmania major, an accepted model for the impact of T‐cell subsets on disease outcome. Here we show that c‐Rel‐deficient (rel−/−) mice infected with L. major displayed dramatically exacerbated leishmaniasis and enhanced parasite burdens. In contrast to WT mice, IFN‐γ and IL‐17 production in response to L. major antigens was severely impaired in rel−/− mice. Reconstitution of Rag1−/− T‐cell deficient mice with rel−/− CD4+ T cells followed by L. major infection demonstrated that c‐Rel‐deficient T cells mount normal Th1 responses and are able to contain the infection. Similarly, Th1 differentiation of naïve CD4+ cells in vitro was normal. Notably, a selective defect in IL‐12 and IL‐23 production was observed in rel−/− DCs compared with their WT counterparts. In conclusion, our data suggest that the expression of c‐Rel in myeloid cells is essential for clearance of L. major and that this c‐Rel‐mediated effect is dominant over the lack of Tregs.