PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation

P21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases with both kinase and scaffolding activity. Here, we investigated the effects of inflammation on PAK1 signaling and its role in colitis-driven carcinogenesis. PAK1 and p-PAK1 (Thr423) were assessed by immunohistochemistry, immunofluorescence, and Western blot. C57BL6/J wildtype mice were treated with a single intraperitoneal TNFα injection. Small intestinal organoids from these mice and from PAK1-KO mice were cultured with TNFα. NF-κB and PPARγ were analyzed upon PAK1 overexpression and silencing for transcriptional/translational regulation. PAK1 expression and activation was increased on the luminal intestinal epithelial surface in inflammatory bowel disease and colitis-associated cancer. PAK1 was phosphorylated upon treatment with IFNγ, IL-1β, and TNFα. In vivo, mice administered with TNFα showed increased p-PAK1 in intestinal villi, which was associated with nuclear p65 and NF-κB activation. p65 nuclear translocation downstream of TNFα was strongly inhibited in PAK1-KO small intestinal organoids. PAK1 overexpression induced a PAK1–p65 interaction as visualized by co-immunoprecipitation, nuclear translocation, and increased NF-κB transactivation, all of which were impeded by kinase-dead PAK1. Moreover, PAK1 overexpression downregulated PPARγ and mesalamine recovered PPARγ through PAK1 inhibition. On the other hand PAK1 silencing inhibited NF-κB, which was recovered using BADGE, a PPARγ antagonist. Altogether these data demonstrate that PAK1 overexpression and activation in inflammation and colitis-associated cancer promote NF-κB activity via suppression of PPARγ in intestinal epithelial cells

Targeting Interleukin-6 for the induction of chimerism-based transplantation tolerance

In den letzten Jahrzehnten entwickelte sich die Organtransplantation zur bevorzugten Behandlungsmethode für das terminale Organversagen. Trotz der kontinuierlichen Verbesserung einiger vielversprechender Protokolle sind Nebenwirkungen wie Infektionen, Nephrotoxizität, Malignität und Stoffwechselstörungen nach wie vor eine erhebliche Belastung für Transplantationspatienten. Aus diesem Grund ist die Etablierung der spenderspezifischen Toleranz eines der höchsten Ziele in der Transplantationsmedizin. In diesem Zusammenhang bietet die Co-Transplantation von Spender-spezifischem Knochenmark (KM) für die Induktion von gemischtem Chimärismus (d.h. Koexistenz von Spender- und Empfängerhämatopoetischen Zellen) eine vielversprechende Methode. Zahlreiche Mausmodelle des gemischten Chimärismus konnten erfolgreich in Primaten Studien überführt werden und auch ihre klinische Relevanz wurde in mehreren Pilotversuchen bei Nierentransplantationspatienten nachgewiesen.^ ^Auf Grund der notwendigen zytotoxischen Vorbehandlung, konnte sich dieser Ansatz jedoch noch nicht als Standardtherapie etablieren. Vor kurzem konnten wir zeigen, dass der Co-Transfer von regulatorischen T Zellen (Tregs) mit Spender KM in Kombination mit Kostimulationsblockade (KB, anti-CD40L und CTLA4-Ig) und mTOR-Hemmung (Rapamycin) eine vielversprechende Methode darbietet, spenderspezifische Toleranz in einem bestrahlungsfreien Protokoll zu erreichen. Der zelluläre Transfer von Tregs wird zurzeit in vielen experimentellen Transplantationsprotokollen getestet, jedoch ist die Umsetzung in die klinische Routine immer noch aufgrund einiger Hindernisse problematisch. Demnach, ist die pharmakologische Immunmodulation zur Anreicherung des endogenen Treg-Repertoires, was in weiterer Folge die KM-Transplantation verbessern soll, eine attraktive Alternative.^ Interleukin-6 (IL-6) zeigte sich in dieser Hinsicht vielversprechend, da es die naive T Zellen Differenzierung zum Th17-Phänotyp fördert und im Gegenzug die Entwicklung der Treg-Linie verhindert. Darüber hinaus ist die Hemmung des IL-6-Signalweges durch das bereits 2010 zugelassene Medikament Tocilizumab (humanisierter IL-6 Rezeptor Antikörper) für die Behandlung von rheumatoider Arthritis und einigen anderen Autoimmunerkrankungen etabliert. Auch im Kontext der Transplantation konnten bereits mehrere Erfolge gezeigt werden. Aufgrund dieser Tatsache untersuchten wir, ob die Blockierung des IL-6-Signalweges die Treg-Differenzierung nach der KM-Transplantation (KMT) fördert und damit die Notwendigkeit einer Treg-Zelltherapie entfällt. Wir konnten zeigen, dass die Blockade von IL-6 das Engraftment von Spender-KM wirksam verbessert und in weiterer Folge zur Induktion eines transienten multi-lineären Chimärismus führt.^ Die Zugabe von anti-IL-6 konnte die Abstoßung von Spender-spezifischen Hauttransplantaten deutlich verzögern und auch ein dauerhaftes Überleben von Spender-spezifischen Herz-Transplantaten wurde gewährleistet. Des Weiteren konnten wir zeigen, dass in diesem bestrahlungsfreien KMT-Modell die anti-IL-6-Therapie die endogene Treg-Expansion fördert, während sie die Aktivierung von Antigen-präsentierenden Zellen (APC) verringert und die Entwicklung von CD8+ T-Effektor-Gedächtnis Zellen (TEM) zeitweise unterdrückt. Zusammenfassend können wir daraus schließen, dass die Blockierung von IL-6 eine wichtige Rolle für nicht-myelosuppressive chimärismus-basierte Toleranzprotokolle darbietet und dadurch eine mögliche Alternative zur Treg-Zelltherapie bereitstellt.Over the last decades, organ transplantation evolved to the treatment strategy of choice for end-stage organ failure. Despite constant improvement of promising drug regimens, several side effects like infections, nephrotoxicity, malignancy and metabolic disorders still remain a substantial burden for transplant patients. Therefore, the establishment of donor-specific tolerance has been one of the utmost goals in transplantation medicine. In this regard, the co-transplantation of allogeneic bone marrow (BM) to induce mixed chimerism (i.e. co-existence of recipient and donor hematopoietic cells within the host) emerged as an attractive approach. Numerous murine mixed chimerism protocols have been successfully translated to non-human primate (NHP) studies and also its clinical relevance has already been proven in several pilot trials performed in renal transplant patients, however its routine clinical application is precluded by the need of myelosuppressive pre-conditioning.^ ^Recently our group could show that the co-transfer of recipient regulatory T cells (Tregs) with donor BM under co-stimulation blockade (CB, anti-CD40L and CTLA4-Ig) and mTOR inhibition (rapamycin) offers promising potential to achieve donor-specific tolerance without myelosuppression in an irradiation-free setting. Adoptive Treg transfer is under investigation in several transplantation protocols, but translation into clinical routine is still impeded by various obstacles. To this end, pharmacological immunomodulation to enrich endogenous Tregs appears to be an attractive alternative strategy. Interleukin-6 (IL-6) was shown to play a pivotal role in this respect because it promotes the differentiation of T cells towards a Th17 phenotype, thereby preventing the conversion into Treg lineage.^ Additionally, inhibition of the IL-6 signaling pathway with Tocilizumab (an anti-IL-6R antibody) is already state of the art for the treatment of rheumatoid arthritis (RA) and other autoimmune diseases. Here we investigated whether blocking IL-6 could promote Treg differentiation after BM transplantation (BMT) and thereby obviate the need for Treg cell therapy. Our results demonstrate that IL-6 blockade effectively improved allogeneic BM engraftment and led to the induction of transient multilineage chimerism. Moreover, the addition of anti-IL-6 significantly prolonged donor skin graft survival and led to indefinite donor cardiac graft survival. Finally, anti-IL-6 therapy promoted endogenous Treg expansion, whilst lowering the activation of antigen presenting cells (APC) and reducing CD8+ effector memory T cell (TEM) generation.^ In summary, our findings could demonstrate that targeting IL-6 may represent a relevant part for non-myelosuppressive chimerism-based tolerance protocols providing an alternative to Treg cell therapy, thereby improving clinical applicability.submitted by Nicolas GranofskyMedizinische Universität Wien, Diss., 201

Combining Adoptive Treg Transfer with Bone Marrow Transplantation for Transplantation Tolerance

Purpose of Review The mixed chimerism approach is an exceptionally potent strategy for the induction of donor-specific tolerance in organ transplantation and so far the only one that was demonstrated to work in the clinical setting. Regulatory T cells (Tregs) have been shown to improve chimerism induction in experimental animal models. This review summarizes the development of innovative BMT protocols using therapeutic Treg transfer for tolerance induction. Recent Findings Treg cell therapy promotes BM engraftment in reduced conditioning protocols in both, mice and non-human primates. In mice, transfer of polyclonal recipient Tregs was sufficient to substitute cytotoxic recipient conditioning. Treg therapy prevented chronic rejection of skin and heart allografts related to tissue-specific antigen disparities, in part by promoting intragraft Treg accumulation. Summary Adoptive Treg transfer is remarkably effective in facilitating BM engraftment in reduced-intensity protocols in mice and non-human primates. Furthermore, it promotes regulatory mechanisms that prevent chronic rejection.(VLID)356997

Transplantation Tolerance through Hematopoietic Chimerism: Progress and Challenges for Clinical Translation

The perception that transplantation of hematopoietic stem cells can confer tolerance to any tissue or organ from the same donor is widely accepted but it has not yet become a treatment option in clinical routine. The reasons for this are multifaceted but can generally be classified into safety and efficacy concerns that also became evident from the results of the first clinical pilot trials. In comparison to standard immunosuppressive therapies, the infection risk associated with the cytotoxic pre-conditioning necessary to allow allogeneic bone marrow engraftment and the risk of developing graft-vs.-host disease (GVHD) constitute the most prohibitive hurdles. However, several approaches have recently been developed at the experimental level to reduce or even overcome the necessity for cytoreductive conditioning, such as costimulation blockade, pro-apoptotic drugs, or Treg therapy. But even in the absence of any hazardous pretreatment, the recipients are exposed to the risk of developing GVHD as long as non-tolerant donor T cells are present. Total lymphoid irradiation and enriching the stem cell graft with facilitating cells emerged as potential strategies to reduce this peril. On the other hand, the long-lasting survival of kidney allografts, seen with transient chimerism in some clinical series, questions the need for durable chimerism for robust tolerance. From a safety point of view, loss of chimerism would indeed be favorable as it eliminates the risk of GVHD, but also complicates the assessment of tolerance. Therefore, other biomarkers are warranted to monitor tolerance and to identify those patients who can safely be weaned off immunosuppression. In addition to these safety concerns, the limited efficacy of the current pilot trials with approximately 40–60% patients becoming tolerant remains an important issue that needs to be resolved. Overall, the road ahead to clinical routine may still be rocky but the first successful long-term patients and progress in pre-clinical research provide encouraging evidence that deliberately inducing tolerance through hematopoietic chimerism might eventually make it from dream to reality

Hybrid resistance to parental bone marrow grafts in nonlethally irradiated mice

Resistance to parental bone marrow (BM) grafts in F1 hybrid recipients is due to natural killer (NK) cellmediated rejection triggered through “missing self” recognition. “Hybrid resistance” has usually been investigated in lethally irradiated F1 recipients in conjunction with pharmacological activation of NK cells. Here, we investigated BMdirected NKcell alloreactivity in settings of reduced conditioning. Nonlethally irradiated (13 Gy) or nonirradiated F1 (C57BL6 BALB/c) recipient mice received titrated doses (520 x 106) of unseparated parental BALB/c BM without pharmacological NK cell activation. BM successfully engrafted in all mice and multilineage donor chimerism persisted longterm (24 weeks), even in the absence of irradiation. Chimerism was associated with the rearrangement of the NKcell receptor repertoire suggestive of reduced reactivity to BALB/c. Chimerism levels were lower after transplantation with parental BALB/c than with syngeneic F1 BM, indicating partial NKmediated rejection of parental BM. Activation of NK cells with polyinosinicpolycytidylic acid sodium salt poly(I:C), reduced parental chimerism in nonirradiated BM recipients but did not prevent hematopoietic stem cell engraftment. In contrast, equal numbers of parental lymph node cells were completely rejected. Hence, hybrid resistance leads to incomplete rejection of parental BM under reduced conditioning settings.(VLID)342372

Anti-Interleukin-6 Promotes Allogeneic Bone Marrow Engraftment and Prolonged Graft Survival in an Irradiation-Free Murine Transplant Model

Transfer of recipient regulatory T cells (Tregs) induces mixed chimerism and tolerance in an irradiation-free bone marrow (BM) transplantation (BMT) model involving short-course co-stimulation blockade and mTOR inhibition. Boosting endogenous Tregs pharmacologically in vivo would be an attractive alternative avoiding the current limitations of performing adoptive cell therapy in the routine clinical setting. Interleukin-6 (IL-6) potently inhibits Treg differentiation and its blockade was shown to increase Treg numbers in vivo. Therefore, we investigated whether IL-6 blockade can replace adoptive Treg transfer in irradiation-free allogeneic BMT. Treatment with anti-IL-6 instead of Treg transfer led to multi-lineage chimerism (persisting for ~12 weeks) in recipients of fully mismatched BM and significantly prolonged donor skin (MST 58 days) and heart (MST > 100 days) graft survival. Endogenous Foxp3+ Tregs expanded in anti-IL-6-treated BMT recipients, while dendritic cell (DC) activation and memory CD8+ T cell development were inhibited. Adding anti-IL-17 to anti-IL-6 treatment increased Treg frequencies, but did not further prolong donor skin graft survival significantly. These results demonstrate that IL-6 blockade promotes BM engraftment and donor graft survival in non-irradiated recipients and might provide an alternative to Treg cell therapy in the clinical setting

DataSheet_1_Chronic CD40L blockade is required for long-term cardiac allograft survival with a clinically relevant CTLA4-Ig dosing regimen.docx

IntroductionIn de-novo kidney transplantation, the CTLA4-Ig fusion protein belatacept is associated with improved graft function but also an increased risk of acute rejection compared to calcineurin inhibitor therapy. The combination with a second costimulation blocker could potentially improve outcome while avoiding calcineurin inhibitor toxicity. The aim of this study was to define the conditions under which the combination of CTLA4-Ig and CD40L blockade leads to rejection-free permanent graft survival in a stringent murine heart transplantation model.MethodsNaïve wild-type or CD40L (CD154) knock-out mice received a fully mismatched BALB/c cardiac allograft. Selected induction and maintenance protocols for CTLA4-Ig and blocking αCD40L monoclonal antibodies (mAB) were investigated. Graft survival, rejection severity and donor-specific antibody (DSA) formation were assessed during a 100-day follow-up period.Results and DiscussionAdministering αCD40L mAb as monotherapy at the time of transplantation significantly prolonged heart allograft survival but did not further improve the outcome when given in addition to chronic CTLA4-Ig therapy (which prolongs graft survival to a median of 22 days). Likewise, chronic αCD40L mAb therapy (0.5mg) combined with perioperative CTLA4-Ig led to rejection in a proportion of mice and extensive histological damage, despite abrogating DSA formation. Only the permanent interruption of CD40-CD40L signaling by using CD40L-/- recipient mice or by chronic αCD40L administration synergized with chronic CTLA4-Ig to achieve long-term allograft survival with preserved histological graft integrity in all recipients without DSA formation. The combination of α-CD40L and CTLA4-Ig works most effectively when both therapeutics are administered chronically.</p