Annette Erhardt

CCR6 Recruits Regulatory T Cells and Th17 Cells to the Kidney in Glomerulonephritis

T cells recruited to the kidney contribute to tissue damage in crescentic and proliferative glomerulonephritides. Chemokines and their receptors regulate T cell trafficking, but the expression profile and functional importance of chemokine receptors for renal CD4+ T cell subsets are incompletely understood. In this study, we observed that renal FoxP3+CD4+ regulatory T cells (Tregs) and IL-17-producing CD4+ T (Th17) cells express the chemokine receptor CCR6, whereas IFNgamma-producing Th1 cells are CCR6-. Induction of experimental glomerulonephritis (nephrotoxic nephritis) in mice resulted in upregulation of the only CCR6 ligand, CCL20, followed by T cell recruitment, renal tissue injury, albuminuria, and loss of renal function. CCR6 deficiency aggravated renal injury and increased mortality (from uremia) among nephritic mice. Compared with wild-type (WT) mice, CCR6 deficiency reduced infiltration of Tregs and Th17 cells but did not affect recruitment of Th1 cells in the setting of glomerulonephritis. Adoptive transfer of WT but not CCR6-deficient Tregs attenuated morphologic and functional renal injury in nephritic mice. Furthermore, reconstitution with WT Tregs protected CCR6-/- mice from aggravated nephritis. Taken together, these data suggest that CCR6 mediates renal recruitment of both Tregs and Th17 cells and that the reduction of anti-inflammatory Tregs in the presence of a fully functional Th1 response aggravates experimental glomerulonephritis.

CXCR3 Deficiency Exacerbates Liver Disease and Abrogates Tolerance in a Mouse Model of Immune-Mediated Hepatitis

The chemokine receptor CXCR3 is preferentially expressed by Th1 cells and critically involved in their recruitment to inflamed tissue. In a mouse model of immune-mediated liver injury inducible by Con A, we investigated the role of CXCR3 in acute IFN-γ–mediated hepatitis as well as in tolerance induction, which has been shown to depend on IL-10–producing CD4+CD25+Foxp3+ regulatory T cells (Tregs). Induction of Con A hepatitis resulted in increased intrahepatic expression of the CXCR3 ligands CXCL9, CXCL10, and CXCL11. CXCR3−/− mice developed a more severe liver injury with higher plasma transaminase activities and a more pronounced Th1/Th17 response compared with wild-type (wt) animals upon Con A injection. Moreover, CXCR3−/− mice did not establish tolerance upon Con A restimulation, although Tregs from CXCR3−/− mice were still suppressive in an in vitro suppression assay. Instead, Tregs failed to accumulate in livers of CXCR3−/− mice upon Con A restimulation in contrast to those from wt animals. Con A-tolerant wt mice harbored significantly increased numbers of intrahepatic CXCR3+T-bet+ Tregs that produced IL-10 compared with nontolerant animals. IFN-γ deficiency or anti–IFN-γ Ab treatment demonstrated that conversion to CXCR3+T-bet+ Tregs depended on a Th1 response. Accordingly, in an immunotherapeutic approach, CD4+CD25+Foxp3+ Tregs from Con A-pretreated CXCR3-deficient mice failed to protect against Con A-induced hepatitis, whereas Tregs from Con A-tolerant wt mice allowed CXCR3-deficient mice to recover from Con A hepatitis. In summary, CXCR3+T-bet+IL-10+ Tregs are generated in the liver in dependence of IFN-γ, then disseminated into the organism and specifically migrate into the liver, where they limit immune-mediated liver damage.

Acetaminophen and pregnancy: short- and long-term consequences for mother and child

Counter-intuitively, over-the-counter medication is commonly taken by pregnant women. In this context, acetaminophen (APAP, e.g. Paracetamol, Tylenol) is generally recommended by physicians to treat fever and pain during pregnancy. Thus, APAP ranks at the top of the list of medications taken prenatally. Insights on an increased risk for pregnancy complications such as miscarriage, stillbirth, preterm birth or fetal malformations upon APAP exposure are rather ambiguous. However, emerging evidence arising from human trials clearly reveals a significant correlation between APAP use during pregnancy and an increased risk for the development of asthma in children later in life. Pathways through which APAP increases this risk are still elusive. APAP can be liver toxic and since APAP appears to freely cross the placenta, therapeutic and certainly toxic doses could not only affect maternal, but also fetal hepatocytes. It is noteworthy that during fetal development, the liver transiently functions as the main hematopoietic organ. We here review the effect of APAP on metabolic and immunological parameters in pregnant women and on fetal development and immune ontogeny in order to delineate novel, putative and to date underrated pathways through which APAP use during pregnancy can impair maternal, fetal and long term childrens health. We conclude that future studies are urgently needed to reconsider the safety and dosage of APAP during pregnancy and - based on the advances made in the field of reproduction as well as APAP metabolism - we propose pathways, which should be addressed in future research and clinical endeavors.

Down-regulation of the De-ubiquitinating Enzyme Ubiquitin-specific Protease 2 Contributes to Tumor Necrosis Factor-α-induced Hepatocyte Survival

Tumor necrosis factor-α (TNFα) stimulation of hepatocytes induces either cell survival or apoptosis, which seems to be regulated by the ubiquitin-proteasome system. Here we investigated the role of TNFα-induced down-modulation of the de-ubiquitinating enzyme USP2 for hepatocyte survival. Inhibition of hepatocyte apoptosis by pre-treatment with TNFα (TNFα tolerance) was analyzed in the mouse model of galactosamine/TNFα-induced liver injury and in actinomycin D/TNFα-treated primary mouse hepatocytes. The role of USP2 for TNFα-induced hepatocyte survival was studied using small interference RNA or an expression clone. Injection of mice or preincubation of hepatocytes with TNFα caused a rapid down-regulation of hepatic USP2–41kD, the predominant USP2 isoform in the liver. In vitro an artificial knockdown of USP2 inhibited actinomycin D/TNFα-induced hepatocyte apoptosis, which was associated with elevated levels of the anti-apoptotic protein c-FlipL/S and a concomitant decrease of cellular levels of the ubiquitinligase Itch, a negative regulator of c-Flip. USP2–41kD overexpression abrogated TNFα tolerance in vitro, prevented accumulation of c-FlipL/S and resulted in elevated levels of Itch. Accordingly, c-FlipL/S protein levels were elevated in livers of TNFα-tolerant mice, which correlated to a switch from JNK and ERK to p38 signaling after galactosamine/TNF re-challenge. Our results indicate that TNFα-induced USP2 down-regulation is an effective cytoprotective mechanism in hepatocytes. Hence, USP2 could be a novel pharmacological target, and specific USP2 inhibitors might be potential candidates for the treatment of inflammation-related apoptotic liver damage.

The neuropeptide calcitonin gene-related peptide (CGRP) prevents inflammatory liver injury in mice.

BACKGROUND/AIMS Calcitonin gene-related peptide (CGRP) is a potent vasodilator and supposed to be responsible for neurogenic inflammation involved in migraine. Its role in inflammatory diseases of other organs is controversial and poorly investigated regarding liver inflammation, although the organ is innervated by CGRP containing primary sensory nerve fibers. METHODS Male Balb/c and IL-10(-/-) mice were pretreated with either alphaCGRP or the CGRP receptor antagonists CGRP(8-37) or BIBN4096BS. Immune-mediated liver injury was induced by administration of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNFalpha) to galactosamine (GalN)-sensitized mice and evaluated by serum transaminase activities and cytokine levels. Furthermore, intrahepatic CGRP receptor expression and hepatic CGRP concentrations were examined. RESULTS CGRP receptor 1 was expressed by immune cells and hepatocytes in human and murine liver. During liver injury CGRP receptor expression was increased whereas hepatic CGRP concentrations concomitantly decreased. While CGRP receptor antagonists failed to affect liver damage, pretreatment with alphaCGRP protected mice from GalN/LPS-induced liver injury by suppression of the pro-inflammatory cytokine response independently from IL-10 but related to the induction of the transcriptional repressor inducible cAMP early repressor (ICER). In contrast, alphaCGRP failed to protect against GalN/TNFalpha-induced liver failure. CONCLUSION In the liver, CGRP exerts anti-inflammatory properties, which are characterized by a reduced production of pro-inflammatory cytokines.

Induction of heme oxygenase 1 prevents progression of liver fibrosis in Mdr2 knockout mice.

Induction or overexpression of the heme‐degrading enzyme, heme oxygenase 1 (HO‐1), has been shown to protect mice from liver damage induced by acute inflammation. We have investigated the effects of HO‐1 induction in a mouse model of chronic liver inflammation and fibrogenesis with progression to hepatocellular carcinoma (HCC) (Mdr2ko; FVB.129P2‐Abcb4tm1Bor). HO‐1 was induced in vivo by treatment with cobalt protoporphyrin IX, starting at week 5 or 12 of mice lifespan, and continued for 7 weeks. Our results showed that HO‐1 induction reduced liver damage and chronic inflammation by regulating immune cell infiltration or proliferation as well as tumor necrosis factor receptor signaling. Fibrosis progression was significantly reduced by HO‐1 induction in mice with mild, as well as established, portal and lobular fibrosis. HO‐1 induction significantly suppressed hepatic stellate cell activation. During established fibrosis, HO‐1 induction was able to revert portal inflammation and fibrosis below levels observed at the start of treatment. Moreover, hepatocellular proliferation and signs of dysplasia were decreased after HO‐1 induction. Conclusion: Induction of HO‐1 interferes with chronic inflammation and fibrogenesis and, in consequence, might delay progression to HCC. (HEPATOLOGY 2012;)

Hepatocytes Contribute to Immune Regulation in the Liver by Activation of the Notch Signaling Pathway in T Cells

The “liver tolerance effect” has been attributed to a unique potential of liver-resident nonprofessional APCs including hepatocytes (HCs) to suppress T cell responses. The exact molecular mechanism of T cell suppression by liver APCs is still largely unknown. In mice, IL-10–dependent T cell suppression is observed after Th1-mediated hepatitis induced by Con A. In this study, we show that HCs, particularly those from regenerating livers of Con A–pretreated mice, induced a regulatory phenotype in naive CD4+ T cells in vitro. Using reporter mice, we observed that these T regulatory cells released substantial amounts of IL-10, produced IFN-γ, failed to express Foxp3, but suppressed proliferation of responder T cells upon restimulation with anti-CD3 mAb. Hence, these regulatory cells feature a similar phenotype as the recently described IL-10–producing Th1 cells, which are generated upon activation of Notch signaling. Indeed, inhibition of γ-secretase and a disintegrin and metalloproteinase 17 but not a disintegrin and metalloproteinase 10, respectively, which blocked Notch activation, prevented IL-10 secretion. HCs from Con A–pretreated mice showed enhanced expression of the Notch ligand Jagged1 and significantly increased receptor density of Notch1 on CD4+ T cells. However, HCs from Con A–pretreated IFN regulatory factor 1−/− mice, which cannot respond to IFN-γ, as well as those from IFN-γ−/− mice failed to augment IL-10 production by CD4+ T cells. In conclusion, it seems that HCs fine-tune liver inflammation by upregulation of Jagged1 and activation of Notch signaling in Th1 cells. This mechanism might be of particular importance in the regenerating liver subsequent to Th1-mediated hepatitis.

Regulatory T Cell–Derived IL-10 Ameliorates Crescentic GN

Regulatory T cells (Tregs) exert their immunosuppressive activity through several immunoregulatory mechanisms, including the production of anti-inflammatory cytokines such as IL-10. Although several studies suggest a role for Tregs in modulating crescentic GN, the underlying mechanisms are not well understood. Here, using IL-10 reporter mice, we detected IL-10-producing Foxp3(+) T cells in the kidney, blood, and secondary lymphoid tissue in a mouse model of crescentic GN. Specific inactivation of Il10 in Foxp3(+) Tregs eliminated the ability of these cells to suppress renal and systemic production of IFNγ and IL-17; these IL-10-deficient Tregs lost their capacity to attenuate renal tissue injury. These data highlight the suppressive functions of Tregs in crescentic GN and suggest the importance of Treg-derived IL-10 in ameliorating disease severity and in modulating both the Th1 and most notably Th17 immune response.

Inhibitor of Apoptosis Proteins as Novel Targets in Inflammatory Processes

Objective—Inhibitor of apoptosis proteins (IAPs), such as X-linked or cellular IAP 1/2 (XIAP, cIAP1/2), are important regulators of apoptosis. IAP antagonists are currently under clinical investigation as anticancer agents. Interestingly, IAPs participate in the inflammation-associated TNF receptor signaling complex and regulate NF&kgr;B signaling. This raises the question about the role of IAPs in inflammation. Here, we investigated the anti-inflammatory potential of IAP inhibitors and the role of IAPs in inflammatory processes of endothelial cells. Methods and Results—In mice, the small molecule IAP antagonist A-4.10099.1 (ABT) suppressed antigen-induced arthritis, leukocyte infiltration in concanavalin A-evoked liver injury, and leukocyte transmigration in the TNF&agr;-activated cremaster muscle. In vitro, we observed an attenuation of leukocyte–endothelial cell interaction by downregulation of the intercellular adhesion molecule-1. ABT did not impair NF&kgr;B signaling but decreased the TNF&agr;-induced activation of the TGF-&bgr;–activated kinase 1, p38, and c-Jun N-terminal kinase. These effects are based on the proteasomal degradation of cIAP1/2 accompanied by an altered ratio of the levels of membrane-localized TNF receptor-associated factors 2 and 5. Conclusion—Our results reveal IAP antagonism as a profound anti-inflammatory principle in vivo and highlight IAPs as important regulators of inflammatory processes in endothelial cells.

Tolerance induction in response to liver inflammation.

Background/Aims: The liver plays an important role in immunological tolerance due to its anatomical location, as it links the gastrointestinal tract and the systemic venous circulation. Therefore, immune reactions against dietary or bacterial antigens from the gut have to be avoided. However, immune responses resulting in elimination of harmful hepatotrophic pathogens have to be induced. We investigated mechanisms of tolerance induction in response to liver inflammation in a murine model of immune-mediated liver injury. Methods: Liver damage was induced by injection of the plant lectin concanavalin A (ConA). Cytokine levels were measured in plasma and liver tissue. The frequencies of intrahepatic and splenic cell subsets were measured by FACS analyses. Results: ConA hepatitis was mediated by activation of CD4+ T cells, NKT cells and Kupffer cells releasing IFN-γ and TNF-α. Tolerance developed towards ConA rechallenge within 8 days, lasted for several weeks and was characterized by significantly reduced plasma transaminase activities, decreased Th1/Th17 responses and an increased IL-10 release, the latter being produced by CD4+CD25+FoxP3+ regulatory T cells and Kupffer cells. Moreover, regulatory T cells from ConA-tolerant mice displayed a higher immunosuppressive potential in vitro and in vivo compared to those from non-tolerant animals. Interestingly, ConA hepatitis was aggravated in CCR5–/– and CXCR3–/– mice. Conclusion: These results suggest that ConA tolerance is mediated by induced IL10+ regulatory T cells, probably trafficking into the liver depending on the IFN-γ-inducible chemokine receptors CCR5 and CXCR3.