Projects 2015 – 2019
Checkpoint A: Switch from pro- to anti-inflammatory cytokine response
It is known that inflammation is based in subsequent waves of white blood cell infiltration guiding the onset, peak and resolution of inflammation. The early phase of the inflammatory response is dominated by cells named neutrophils (neutrophil granulocytes belong to white blood cells), its later phase is characterized by scavenger cells so-called macrophages and particularly eosinophils (a subgroup of leucocytes, the white blood cells) during the resolution phase of inflammation. It’s tempting to speculate that effective resolution of inflammation has to overcome pro-inflammatory cytokine production by macrophages. Macrophages belong to the key cytokine producers in the body. And cytokines are regulatory proteins which trigger the inflammation process. The switch from the pro- to the anti-inflammatory cytokine responses seems to be one level of decision if inflammation becomes chronic or not. So it is important to decipher the cellular mechanisms which control this switch.
Rheumatoid arthritis (RA) is the most severe chronic inflammatory joint disease. It is characterized by a high level of chronicity and the failure to spontaneously resolve inflammation despite absence of the inflammatory trigger. Ineffective resolution is therefore a major clinical challenge and unmet need in understanding arthritis. To date, the factors leading to resolution of arthritis remain obscure.
This project aims to define a cellular and molecular pathway, which fosters the resolution of inflammatory arthritis. We will therefore revisit a part of the immune response, which is well known for allergy but largely unexplored in the field of arthritis. We suspect that specific T cells (Th2 cells) alternatively-activated macrophages (AAM) and eosinophils may at the same time foster the resolution of inflammatory diseases like arthritis.
In support of this concept, we show that a robust activation of the Th2-AAM-eosinophil axis mitigates the course of inflammatory arthritis, suggesting that this pathway can impact this severe inflammatory joint disease. We will use these results to characterize cellular and molecular mechanisms in the resolutions process of inflammation in arthritis. In order to reach fast translation of research results into clinical practice, we will analyze the Th2-AAM-eosinophil axis in patients with true resolution of the disease or where the disease is only suppressed by anti-inflammatory drugs. These analyses will allow us to get a comprehensive view on the mechanisms of resolution in arthritis and potential strategies to foster this process.
Alternatively activated macrophages (AAMs) are associated with tissue remodeling, wound healing and resolution of inflammation. The development of AAMs is critically dependent on the cytokines IL-4 or IL-13 which share similar receptors on the cell surface. IL-4 and IL-13 mainly activate the transcription factor STAT6 which orchestrates the developmental program of AAMs.
In this project we investigate the contribution of AAMs for resolution of inflammation in the lung and intestine. We will analyze possible differences between gene expression profile of cytokine IL-4 and IL-13 induced AAMs model and human samples. Further we will identify STAT6-target genes in macrophages and characterize molecular by genome-wide Chromatin immunoprecipitation analysis. By using a constitutively active version of STAT6 we will determine if active STAT6 is sufficiently for AAM differentiation.
After tissue damage and cellular necrosis, alarmins such as IL-1α, IL-33 and HMGB1 cooperatively initiate an inflammatory response via the signalling adaptor molecule MyD88. Normally, this sterile inflammation resolves and tissue repair is initiated. Mechanisms that actively initiate such pro-resolving events are poorly understood, though.
Our preliminary data show that necrotic cell-derived IL-33 contributes not only to necrosis-induced inflammation, but likewise coordinates its resolution and additionally promotes the clearance of necrotic tissue.
During the proposed project, we plan to delineate the dual role of IL-33 in the response to tissue injury. As our preliminary data indicate that the pro-resolving properties of IL-33 involve the proliferation and differentiation of alternatively activated macrophages (AAMs), we will put special emphasis on the impact of this cytokine on macrophage differentiation and function. Moreover, we seek to understand the molecular mechanisms underlying the divergent pro- and anti-inflammatory effects of IL-33 and to identify potential targets for the diagnosis and therapy of tissue injury-related diseases.
Helicobacter pylori is a paradigm agent for persistent bacterial infection and chronic inflammation in humans. The chronicity of inflammation during H. pylori infection is related to the bacterial manipulation of regulatory cytokines involved in the resolution of inflammation. In general, regulated actions of pro- and anti-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β) and various interleukins (IL-1β, IL-18 and IL-6), have been implicated in resolution of inflammation and maintaining tissue homeostasis. Hence, changes in the balance of these cytokines influence the resolution of inflammation in addition to the clearance of microbes.
we aim to compare the regulatory factors involved in the resolution of inflammation in H. pylori infection in mice and humans. For a better understanding of chronification and resolution of H. pylori infection provide a three-stage is composed of i) investigation of pro- and anti-inflammatory cytokine production, ii) receptor dynamic and iii) the involvement of regulatory molecules. In addition, a screening of a mutant library of H. pylori will identify the bacterial factors that manipulate the molecular checkpoints deciding resolution in this infection.
Preliminary results showed for IL-36, a member of the IL-1-family, that it plays a crucial role in inflammatory processes of psoriasis: mice without IL-36 receptor antagonist (IL-36Ra) show a severe skin disease similar to patients with functional mutations in IL-36RN. The latter have so-called pustular psoriasis (GPP) – a rare, multisystemic, and potentially life-threatening form of psoriasis with diffuse, erythematous skin inflammation is characterized by epidermal pustules accumulating numerous neutrophils. Disease models suggest that mutations in IL36RN lead to an imbalance in the IL-36 pathway resulting in an enhancement of IL-36 signaling and pro-inflammatory mediators. We could show that both, IL-36 signaling and IL-36Ra, are expressed in inflamed arthritic tissues. Fibroblasts respond to IL-36 through MAPK activation. This leads to the production of neutrophil attractant cytokines/ chemokines and fibroblast proliferation. However, the mechanisms leading to resolution of psoriatic diseases remain to be clarified.
We hypothesize that deregulation of different members of the IL-36 pathway accounts for the pathogenic processes leading to the accumulation of neutrophils in inflamed tissue of skin and joint disease. The process of resolution fails and inflammation perpetuates. The molecular mechanisms controlling the switch from pro- to anti-inflammatory cytokine responses during the resolution phase of inflammation will be studied by addressing the following aims: Firstly, we will identify mutations in further members of the IL-36 pathway in GPP and other forms of psoriasis. Secondly, the response to IL-36 family members with regard to recruitment of neutrophils and resolution of disease in psoriatic arthritis will be examined. Thirdly, we will evaluate further genes and pathways for GPP and their relevance for psoriatic disease. So far, no cure of chronic inflammatory disease exists. These approaches will provide the basis for a new understanding for this severe inflammatory skin and joint disease and for developing new therapeutic strategies targeting chronic inflammation.
Stefan Haskamp Madelaine Hahn
The obligate intracellular, Gram-negative bacterium Coxiella burnetii is the causative agent of the zoonotic disease Q-fever. Q-fever is often a mild self-limited flu-like illness, but can develop into an interstitial pneumonia or hepatitis. Furthermore, the infection may also become chronic. Chronic Q-fever is characterized by bacterial persistence in macrophages, increased IL-10 production, atypical M2 polarization of infected macrophages, and lack of granuloma formation. Until now the host and pathogen determinants of resolution or chronicity in C. burnetii infection have not been well defined.
We aim to identify how host and pathogen determinants influence cell death, cytokine secretion, and intracellular signaling in C. burnetii infection. Thus, by investigating both host and pathogen factors we aim to clarify how C. burnetii infection resolves or develops into chronic inflammation. This information may help to develop new urgently needed treatment strategies for chronic Q-fever.
Many autoimmune diseases are characterized by a simultaneous loss of humoral tolerance and chronic inflammation. IgG autoantibodies in the form of immune complexes or as cytotoxic antibodies play a crucial role in tissue inflammation by triggering pro-inflammatory effector pathways including the release of cytokines, the activation of the complement pathway and activation of innate immune effector cells via binding to Fc-receptors widely expressed on these immune cell subsets. Apart from this pro-inflammatory activity of IgG autoantibodies, polyclonal IgG preparations (intravenous immunoglobulins or IVIg) pooled from thousands of healthy donors are effective in resolving acute and chronic inflammation. Although widely used in the clinic, the mechanism underlying this anti-inflammatory activity of IVIg preparations under therapeutic conditions remains largely unknown, as the majority of previous studies were exclusively using preventive treatment schemes.
Thus, the central aim of this project is to define the cellular and molecular components of the immunomodulatory pathway underlying IVIg mediated resolution of established inflammation in several preclinical and clinical settings of chronic inflammation. These studies may not only help to understand the mode of action of a widely used and very successful anti-inflammatory drug, but to decipher the basic components and the biology of the naturally occurring pathway responsible for the resolution of inflammation and its impairment in chronic autoimmune disease.
Immunological processes within the scope of type 2 immune responses leads to resolution of inflammation and tissue repair or foster progression of disease, are largely unknown. Recently, distinct populations of innate lymphoid cells (ILC) were introduced as important mediators of both protective immunity and autoimmune tissue destruction. Recently, distinct populations of innate lymphoid cells (ILC) were introduced as important mediators of both protective immunity and autoimmune tissue destruction. Type 2 innate lymphoid cells (ILC2) are considered being the particular ILC subset that may drive central features of type 2 responses in vivo. ILC2 are particularly enriched in organs with epithelial surfaces such as the lung. Their activation via epithelial derived cytokines (IL-33, IL-25 and TSLP) is a key aspect of their immunological functions.
ILC2 have been shown to express receptors for pro-resolving lipid mediators, secrete tissue repair factors like amphiregulin and regulate via constitutive and inducible production of IL-5, IL-9 and IL-13 steady state and inducible numbers of eosinophils and alternatively activated macrophages. In contrast, dysregulated ILC2 activation has been strongly implicated in chronic inflammatory immune responses that contribute to organ destruction and tissue remodeling. Overall, these data clearly suggest that a detailed understanding of cellular and molecular pathways that regulate ILC2 functions may be particularly important for a broad understanding of inflammatory processes at mucosal surfaces and other parts of the body. However, how factors of the immune system control ILC2 and their effector functions is largely unknown.
In preliminary studies, we identified by the IL-12 related immunoregulatory cytokine IL-27 as important modulator of ILC2 functions in vitro and in vivo in the lung. Therefore, the overall goal of this proposed project is to investigate, how IL-27 regulates the critical balance between the tissue protective and the proinflammatory functions of ILC2 at mucosal surfaces. In addition, we plan to correlate basic research data to immunological situation of patients with lung diseases and to evaluate the potential of ILC2 directed therapies for the treatment of patients with acute and chronic lung diseases.
Checkpoint B: Blockade of pro-inflammatory lymphocyte activation
The blockade of pro-inflammatory lymphocyte activation, refelcting a break of immune tolerance, may precede the onset of chronic inflammatory diseases, as observed in rheumatoid arthritis. Such sequence of pathologic events appears to be absent in other forms of chronic inflammatory diseases such as Crohn’s disease. Chronic inflammatory diseases are characterized by a consistent activation of the adaptive immune system and the appearance of lymphocytes, cellular blood components which include T cells, B cells and natural killer cells, in the inflammatory lesions.
The scientists in Erlangen hypothesize that the pattern of T cell activation in inflammatory disease may emerge as a crucial checkpoint controlling either resolution of inflammation or its chronification. It can be assumed a tight cross talk of T cells with the macrophage polarization and other regulatory innate processes as described above as well with resistant tissue cells that together will determine the outcome of an inflammatory process.
IL-9 production was initially associated with a Th2-like phenotype but later a specific T cell subset responsible for IL-9 production (Th9 cells) was identified. IL-9 gene expression in T cells is regulated by the transcription factors STAT1/IRF1, IRF4 and the Ets-transcription factor PU.1. In this project, we wish to analyze the role of IL-9 producing T cells in driving altered resolution of chronic mucosal inflammation.
Preliminary results demonstrated that IL-9 and PU.1+ Th9 cells are highly expressed in patients with ulcerative colitis as well as in acute oxazolone-induced experimental colitis. Furthermore, oxazolone-induced colitis could be prevented by anti-IL-9 antibody therapy, IL-9 deficiency or selective inactivation of PU.1 in T cells. In this project we will analyze the role of IL-9/ Th9 cells during transition of acute into chronic colitis. Our studies aim at the further characterization of the functional role of Th9 cells during impaired resolution of inflammation in IBD patients and experimental colitis. Our studies will lead to new insights into the molecular pathophysiology of IBD and may result in new concepts for clinical therapy.
The objective of this project is the elucidation of the mode of action of indoleamin-2,3-dioxygenase (IDO) in soluble CD83 (sCD83) mediated immune modulatory processes. In the long term we aim to develop new therapies for inflammatory autoimmune disorders such as rheumatoid arthritis (RA). Previously we could show that sCD83 potently modulates immune responses in vitro as well as in vivo, thereby inducing regulatory T cells in an IDO dependent manner, which leads to the resolution of inflammation. However, the precise underlying molecular and cellular mechanisms are unknown and will therefore be investigated using two different animal models of inflammatory arthritis.
We hypothesize that sCD83 improves the resolution of arthritis through IDO mediated mechanisms, whereas the absence of IDO will facilitate the chronification of arthritis. We further suggest that sCD83 does not just block inflammation by inhibiting inflammatory mediators such as pro-inflammatory cytokines, but actively induces intrinsic mechanisms, via IDO and Tregs, leading to the resolution of inflammatory processes. Regarding the translational importance of CD83 in the human setting, we could previously show that in the synovial fluid of RA patients elevated levels of sCD83 are present. Thus, sCD83 may play a role in modulating immune responses within the rheumatoid joint. Thus, sCD83 levels could represent a useful marker in different stages of RA including the resolution phase. This translational aspect will be further investigated within this proposal, and we have access to samples from the RETRO study, which specifically aims to study resolution of inflammation in a cohort of 100 RA patients subjected to tapering and stopping of medication to distinguish resolution from suppression of disease.
Regulatory T cells (Treg) play an important role in the maintenance of immune tolerance to self antigens and are involved in modulating immune responses to promote resolution of inflammation. In this project we will characterize the immune regulatory function of largley unknown population suppress T cells, a novel subset of TCRαβ+ CD4– CD8– double-negative (DN) T cells, in human system. In preliminary work we showed that human DN T cells have a immense activity and inhibit proliferation and functionality of effector cells.
In this project we will analyze through which specific key molecules and signaling pathways DN T cells suppress effector cells and how far they could modulated these mechansims. Finally, we will analyze whether human DN T cells offer an in vivo regulatory function and affect Graft-versus-Host disease (GvHD) in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for promoting the resolution of acute and/or chronic inflammatory graft-versus-host reactions.
Inner, mucosal surfaces are often affected by chronic, T cell–mediated disease states as in the case of inflammatory bowel disease (IBD) or chronic lung inflammation such as asthma and chronic obstructive pulmonary disease (COPD). Consecutively therapeutic strategies targeting these mediators of inflammation (e. g. anti-TNF-á) were successfully introduced into the routine therapy. However, the minority of IBD patients remains in stable remission while the majority is unable to resolve mucosal inflammation indicating alternative pathogenic mechanisms.
T helper (Th) cells are central regulators in mucosal inflammatory processes during IBD. According a series of experimental data in murine colitis and in human IBD tissue samples suggesting Interleukin 17a (IL-17a)-produced CD4+ T cell so-called Th17 cells within the T helper cell population a functional role during colitis pathogenesis. In line with a dominant role of Th17 cells as drivers of mucosal inflammation, we provide evidence that mice with inactivated Basic leucine zipper transcription factor, ATF-like (Batf) lack Th17 cells and fail to mount T cell-driven intestinal inflammation. In contrast to our studies show that Batf expressed T cells assume mucosal protective functions and so contribute to resolution of mucosal inflammation.
In the center of this project proposal are studies designed to systematically deconstruct the temporal-, spatial- and context-dependent composition of the Batf-dependent vs. -independent T cell intrinsic molecular program which drives the resolution of mucosal inflammation. The obtained results will systematically transferred to clinical human situation. These data may help to design novel therapeutic strategies that redirect colitis mediating T cells to an inflammation-resolving, regulatory phenotype in vivo.
Siglecs (Sialic-acid-binding immunoglobulin-like lectins) are a family of receptors on immune cells, which bind to their ligands sialic acids and have mainly inhibitory functions. We could previously show that CD22 is a functional inhibitory receptor on B cells, while Siglec-G is an inhibitory receptor particularly for the subpopulation of B-1 cells. The deficiency of both Siglecs together leads to autoimmunity in mice. Based on their functional role in inhibiting immune cells of the innate and adaptive immune system, Siglec proteins may play an important role in the resolution process during inflammation. Siglec-H and Siglec-15 are recently characterized members of this family. Siglec-H is expressed in a restricted fashion on plasmacytoid DCs (pDCs), while Siglec-15 is found on macrophages, conventional DCs and osteoclasts. Initial studies show that Siglec-15 seems to be functionally important for osteoclast differentiation, while Siglec-H inhibits IFNα production in pDCs. This suggests that Siglec-H normally leads to a resolution of inflammation triggered by virus infection, but without this Siglec chronicity may result.
In this project we want to further examine the role of Siglec-H in autoimmune disease as Systemic Lupus Erythematosis (SLE) and rheumatoid Arthritis (RA). Further we will be determined how loss of Siglec-15 affects osteoclast functions. Furthermore, we aim to develop Siglec-H- and Siglec-15-specific high affinity synthetic sialic acid ligands to target these Siglecs in autoimmune diseases. Such a strategy has already been used for other Siglecs expressed on other immune cells.
Checkpoint C: Fostering of tissue remodeling by cell death and tissue repair mechanisms
Resolution of inflammation requires the controlled removal of immune cells to debunk the inflammatory infiltrate. In addition, resolution of inflammation is accompanied by tissue responses entailing the repair and/or the replacement of damaged tissue. Effective resolution therefore has to allow for the death or evasion of immune cells and for a directed response by resident tissue to reconstitute the tissue architecture at the site of former inflammation. These tissue responses do not only have a limit inflammation but they may also exert intrinsic anti-inflammatory effects. How cell death and reprogramming of resident tissue cells and immune cells contribute to the resolution of inflammation and repair will be investigated within the third step of decision.
We characterize the retinoid-related orphan receptor α (RORα), a member of the superfamily of nuclear receptors, as a novel regulator of inflammation and tissue remodeling. Our preliminary data demonstrate RORα is induced by pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) in early stages of normal wound healing in monocytes and fibroblasts. The upregulation of RORα stimulates the release of profibrotic cytokines from monocytes and promotes collagen release from fibroblasts. Increasing levels of TGF-β inhibit the expression of RORα in later stages of physiological wound healing. We further demonstrate that the downregulation of RORα counteracts the activation of monocytic cells and fibroblasts and allows resolution of inflammation and normalization of the release of extracellular matrix, thus terminating the wound healing response. In contrast a defective inhibition of RORα leads to persistent tissue response in fibrotic diseases.
In our project, we aim to extend our preliminary data and to further characterize RORα as a checkpoint for the regulation of inflammation and tissue responses. We plan to identify the molecular regulation of RORα in the context of inflammation and fibrosis, to characterize the intracellular pathways regulated by RORα and to evaluate RORα as potential target for the treatment of chronic inflammatory diseases with pathologic tissue responses.
Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn´s disease, are disabling illnesses which are distinguished by progressive destructive character. The pathogenesis of these chronically relapsing disorders has not been fully clarified, and therapeutic options for the resolution of intestinal inflammation are still limited.
However, it´s well established that CD4+ T cells are key players influencing the initiation and maintenance of chronic inflammation in the gut. TNFR2-signaling and CASP8 are central regulators in multiple T cell fate decisions including different forms of cell death. Published data suggest that CASP8 can interact with TNFR2 signaling in T lymphocytes and both molecules can promote pro- as well as anti-inflammatory effector functions depending on the cellular and molecular context.
Our own data have provided evidence that TNFR2-signaling and CASP8 can guide molecular mechanisms promoting the resolution of inflammation in the gut. Further we have recently demonstrated that TNFR2 ex-pressing mucosal T cells are the principal targets of therapeutically efficient anti-TNF antibodies in IBD patients.
The project combines investigations in experimental colitis models and primary human intestinal cell to achieve a better understanding about molecular and cellular associations and functions of mucodal TNFR2+CD4+ T cells as well CASP8 and TNFR2 signaling.
The use murine models allow to analyze the cellular/molecular context dependent roles of TNFR2 and CASP8 as central molecules in perpetuation and resolution of inflammation. In the furter project part we aim to characterize the properties of human TNFR2+CD4+ mucosal T cells in the context of anti-TNF antibody treatment in IBD patients and a particular focus will be put on molecular mechanisms causing resistance of mucosal TNFR2+CD4+ T cells to anti-TNF therapy.
Our project aims to contribute to the development of novel therapeutic options for patients suffering from IBD.
Activated neutrophil granulocytes release high amounts of pro-inflammatory mediators to the site of inflammation, NETs form large aggregates (aggNETs) that trap and sequester their pro-inflammatory content by inherent serine proteases. How these self-reinforcing processes will be controlled so that inflammation will not be out of controll, chronic and attack the body is the central question of this project.
Activated granulocytes form neutrophil-extracellular-traps (NETs) which catch and eliminate intruding microorganisms. Recently we showed that NETs resolve inflammation: NETs form large aggregates (aggNETs) that trap and sequester their pro-inflammatory content by inherent serine proteases. Consequently, MSU-induced inflammation spontaneously resolves within a few days, although the inflammatory trigger may still be present in situ.
In this project we will elucidate the role of neutrophils in the process of resolution of inflammation by defining a fundamental and conserved mechanism by which the body contains cytokine production and activity at inflammatory sites.
Arginine catabolism by arginase (Arg)1 and 2, which generates urea and ornithine and subsequently leads to the formation of polyamines and collagens, has been linked to cell proliferation and tissue repair, but also to immunosuppression, pathogen survival and organ fibrosis. According to currently prevaling concepts Arg1 is a marker of alternatively activated, regulatory or suppressor macrophages, signifies anti-inflammatory processes and counteracts the activity of inducible nitric oxide (NO) synthase (iNOS, NOS2). NOS2 effect anti-microbial and compete for comon substrate L-Arginin with Arginase 1 and Arginase 2.
recently observed a strikingly improved clinical course of disease the impact of Arginase 1 on chronic epithelial inflammation. The overarching aim of the project is to identify the cellular and microenvironmental parameters that determine the effect of Arg1 and 2 in different phase of inflammation (induction, perpetuation, and resolution). Hereto we will characterize the expression and regulation of arginases and define the isoform- and cell-specific impact of Arg on the development of chronic colitis and CL by analysing the course of the diseases in experimental models. These approaches will set the basis for using Arg1/2 as novel targets for clinical intervention.
According the current knowledge chronic inflammatory diseases (CED) develop due to continously confrontation of mucosal immun system with microbial antigens in intestinal flora. Dysorders in epithelial barrier functions advanced these processes as also may caused by inflammatory response itself.
Resolution of intestinal inflammation during phases of remission in inflammatory bowel disease patients requires epithelial restitution and a re-establishment of robust barrier function. This is a critical step since repair greatly limits the influx of microbial content into the lamina propria and therefore prevents excessive confrontation of the gut immune system with luminal antigens, a presumed driver of IBD pathogenesis. How re-establishment of barrier function is achieved in the presence of an inflammatory environment such as during an inflammatory flare of IBD is currently unknown.
We and others have recently demonstrated a critical role for caspase-8 dependent signaling cascades for intestinal barrier function. Deregulated activation of caspase-8 is associated with excessive epithelial cell apoptosis, barrier dysfunction and persistent intestinal inflammation. Conversely, however, inactivation of caspase-8 is associated with epithelial cell necroptosis, barrier dysfunction and intestinal inflammation. We hypothesize that regulation of caspase-8 within the intestinal epithelium plays an important role in the resolution of gut inflammation by protecting the epithelium from inflammatory cell death and allowing epithelial restitution and barrier re-establishment.
In our project we attempt how intracellular signaling pathways regulate epithelial cell necroptosis during resolution of intestinal inflammation. One of the project aims is to elucidate the regulation and functional role of epithelial necroptosis during resolution of intestinal inflammation. In a second step, we seek to investigate the role of STAT1 in RIP-dependent and independent intestinal epithelial necroptosis. Finally, we will study the impact of TNF neutralization on intestinal epithelial cells of patients and in models of intestinal inflammation.