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B-cell acute lymphoblastic leukaemia (B-ALL) is characterized by the overproduction of lymphoblasts in the bone marrow (BM), and it is the most common cancer in children while being comparatively uncommon in adults. On the other hand, in chronic myeloid leukaemia (CML), 70% of cases are found in patients older than 50 years, making it uncommon in children. All CML cases and up to 3% of paediatric B- ALL (and 25% of adult B-ALL) cases are due to fusion gene BCR-ABL1, which gives rise to the cytoplasmatic, constitutively active oncoprotein, tyrosine kinase BCR-ABL1 through a reciprocal translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase leads to deregulation of different signal transduction pathways such as cell growth, proliferation and cell survival. The role of the bone marrow microenvironment (BMM) can mediate disease initiation (only in mice), progression, therapy resistance, and relapse, as has been increasingly recognized over the last two decades. In general, the BMM is a very complex arrangement of various cell types such as osteoblasts, osteoclasts, endothelial cells, adipocytes, mesenchymal stromal cells, macrophages and several others. In addition, the BMM is composed of multiple chemical and mechanical factors and extra cellular matrix (ECM) proteins which contribute to the BMM’s features influencing leukaemia behaviour. Considering the incidence of B-ALL and CML in children and in adults respectively, we hypothesized that the young and/or an aged BMM might also play a previously unrecognized role in the aggressiveness of B-ALL and CML. We proposed that BM, transduced with BCR-ABL1-expressing retrovirus in the murine transduction/transplantation model of B-ALL, transplanted into young versus old recipient mice would lead to a more aggressive disease in young mice, and similarly CML would be more aggressive in old recipient mice. In close recapitulation with the human incidence, induction of CML led to a significantly shorted survival in old recipient mice. On the other hand, induction of B-ALL showed a shortened survival in young compared to old syngeneic mice, as well as in a xenotransplantation model. Among the highly heterogenous composition of the BMM, we implicate young BM macrophages as a supportive niche for B-ALL cells. The results were found to be mostly due to potential soluble factors differentially secreted from young and old macrophages. Therefore, we hypothesized that the chemokine CXCL13, which has been demonstrated to play a role in B cell migration and act as a diagnostic marker in the cerebrospinal fluid of patients with neuroborreliosis, might be responsible for the observed phenotype. CXCL13 was found to be more highly expressed in healthy and leukaemic young mice as well as in conditioned medium of young macrophages. Using a variety of in vitro experiments, CXCL13 showed to significantly increase the proliferation and the migration of leukaemia cells when exposed to young macrophages, and the phenotype was rescued while using a CXCL13 neutralizing antibody. The CXCL13 role was also confirmed in vivo, since macrophage ablation led to a prolongation of survival in young mice and a reduction of CXCL13 levels. The use of an additional mouse model, leukaemia cells with CXCR5 deficiency, led to a significant prolongation of survival of young mice, confirming the importance of the CXCL13-CXCR5 axis in B-ALL. In line with our murine results, we found that human macrophages and CXCL13 levels were higher in pediatric B-ALL patients than in adults. Consistent with our murine data, the expression level of CXCR5 may act as a prognostic marker in B-ALL, as well as a predictive marker for central nervous system relapse in human B-ALL. The overall findings show that a young BMM, and in particular macrophages, influences B-ALL progression. We specifically identified CXCL13, secreted by young macrophages, as a promoter of proliferation of B-ALL cells, influencing survival in B-ALL via CXCR5. The CXCR5-CXCL13 axis may be relevant in human B-ALL, and higher CXCR5 expression in human B-ALL may act as a predictive marker.
Two main types of methods are used in gene therapy: integrating vectors and nuclease-based genome engineering. Nucleases are site-specific and are efficient for knock-outs, but inefficient at inserting long DNA sequences. Integrating vectors perform this task with high efficiency, but their insertion occurs at random genomic positions. This can result in transformation of target cells, which leads to severe adverse events in a gene therapy context. Thus, it is of great interest to develop novel genome engineering tools that combine the advantages of both technologies. The main focus of this thesis is on generating such a targetable integrating vector.
The integrating vector used in this project is the Sleeping Beauty (SB) transposon, a DNA transposon characterized by high activity across a wide range of cells. The SB transposase was combined with an RNA-guided Cas9 nuclease domain. This nuclease component was meant to direct transposase integration to specific targets defined by RNAs. The SB transposase was fused to cleavage-inactivated Cas9 (dCas9) to tether it to the target sites. In addition, adapter proteins consisting of dCas9 and domains non-covalently interacting with SB transposase or the SB transposon were generated. All constituent domains of these fusion proteins were tested in enzymatic assays and almost all enzymatic activities could be verified.
Combining the fusion protein dCas9-SB100X with a gRNA binding a sequence from the AluY repetitive element resulted in a weak, but statistically significant enrichment around sites bound by the gRNA. This enrichment was ca. 2-fold and occurred within a 300 bp window downstream of target sites, or within the AluY element.
Targeting with adapter proteins and targeting of other targets (L1 elements or single-copy targets) did not result in statistically significant effects. Single-copy targets tested included the HPRT gene and three specifically selected GSH targets that were known to be receptive to SB insertions. The combination with a more sequence-specific transposase mutant also failed to increase specificity to a level allowing targeting of single-copy loci. Genome-wide analysis of insertions however demonstrated, that dCas9-SB100X has a different insertion profile than SB100X, regardless of the gRNA used.
As low efficiency of retargeting is likely a consequence of the high background activity of the SB100X transposase in the fusion constructs, a SB mutant with reduced DNA affinity, SB(C42), was generated. For this mutant, transposition activity was partly dependent on a dCas9 domain being supplied with a multi-copy target gRNA, specifically a 2-fold increase in the presence of a AluY-directed gRNA. Whether using this mutant results in improved targeting remains to be determined.
In a side project, an attempt was made to direct SB insertions to ribosomal DNA by fusing the transposase to a nucleolar protein. This fusion transposase partially localized to nucleoli and insertions catalyzed by this transposase were found to be enriched in nucleolus organizer regions (NORs) and nucleolus-associated domains (NADs).
The aim of a second side project was increasing the ratio between homology-directed repair (HDR) and non-homologous end-joining (NHEJ) at Cas9-mediated double-strand breaks (DSBs). To achieve this, Cas9 was fused to DNA-interacting domains and corresponding binding sequences were fused to the homology donors. While an increased HDR/NHEJ ration could be observed for the fusion proteins, it was not dependent on the presence on the binding sequences in the donor molecules.
Schätzungen zufolge sind weltweit etwa 71 Millionen Menschen chronisch mit dem Hepatitis-C-Virus (HCV) infiziert. Im Jahre 2016 sind rund 400.000 Menschen an einer HCV-bedingten Lebererkrankung gestorben, insbesondere aufgrund der Entwicklung von Leberzirrhose und Lebertumoren. Trotz der großen Unterschiede in den Prävalenzschätzungen und der Qualität der epidemiologischen Daten zeigt die jüngste weltweite Bewertung, dass die virämische Ausbreitung der HCV-Infektion (Prävalenz der HCV-RNA) in den meisten Industrieländern, einschließlich der USA, weniger als 1,0% beträgt (www .cdc.gov / Hepatitis / HCV). In einigen osteuropäischen Ländern wie Lettland (2,2%) oder Russland (3,3%) und bestimmten Ländern in Afrika, Ägypten (6,3%) und Gabun (7,0%) oder im Nahen Osten Syriens (3,0%) ist die Prävalenz bemerkenswert höher. In den USA und den am weitesten entwickelten Ländern gilt die gemeinsame Nutzung von Werkzeugezur Herstellung von Arzneimitteln und zur Injektion von Medikamenten (Nadeln) als die häufigste derzeitige Übertragungsart. Die vorherrschende Übertragungsart in Ländern, in denen die Ausbreitung von HCV-Infektionen im Vergleich zu den Industrieländern höher ist, beruht jedoch auf schlechten Methoden zur Infektionskontrolle und unsicherer Handhabung von Injektionsnadeln.
Wenn die chronische Infektion unbehandelt bleibt, kann sich im fortschreitenden Verlauf eine Zirrhose oder ein hepatozelluläres Karzinom bilden (Alter H. J. und Seef L. B. 2000). Die Doppeltherapie, bei der es sich um eine Kombination aus pegyliertem Interferon-α (PEG IFNα) und Ribavirin (riba) handelt, war in einigen Ländern der Dritten Welt bis vor kurzem der goldene Standard für die Behandlung von Patienten mit chronischer Hepatitis C und hat eine anhaltende virologische Reaktion erzielt. Mit nur 50% der mit HCV-Genotyp 1 infizierten Patienten (der häufigere) im Vergleich zu 80% mit Genotyp 2 oder 3, obwohl sie kostspielig und langwierig sind (z. B. 24-48 Wochen) und zahlreiche harte Nebenwirkungen aufweisen, die schwer zu bekämpfen sind tolerieren (Erklärung der National Institutes of Health Consensus Development Conference: Management von Hepatitis C: 2002 - 10.-12. Juni 2002 2002). Die Identifizierung des JFH1 (japanische fulminante Hepatitis Typ 1) -Isolats wurde in einigen in vitro-Studien zu HCV als wichtiger Durchbruch bei der HCV-Behandlung angesehen. Die Verwendung dieses Isolats führte nachfolgend zu einem besseren Verständnis des HCV-Lebenszyklus und der 3D-Strukturen der viralen Proteine. Basierend auf dieser Erkenntnis konnten die ersten direkt wirkenden antiviralen Mittel (DAAs) entwickelt werden, die spezifisch virale Proteine beeinflussen. Die beiden Proteasehemmer (PI) Telaprevir und Boceprevir hemmen die virale NS3-4A-Protease und wurden 2011 als Kombinationstherapie mit PEG IFNα und Ribavirin zugelassen, was die anhaltende virologische Reaktion auf 67-75% erhöhte (Pawlotsky et al. 2015).
Die Optimierung der gegenwärtigen Arzneimittelregime, die Einschränkung des Problems der Mutationsresistenz, die Gestaltung einer individualisierten Therapie, der Zugang zu diesen therapeutischen antiviralen Arzneimitteln und ihr hoher Preis bleiben weiterhin eine Herausforderung (Pawlotsky 2016; Pawlotsky et al. 2015; Sarrazin 2016). Die Entwicklung eines Impfstoffs wird jedoch als größte Herausforderung für die weltweite Kontrolle von HCV angesehen (Bukh 2016). Aus diesem Grund ist es wichtig, weiterhin mehr über den HCV-Lebenszyklus und die Faktoren zu erfahren, die sich auf die Replikation und den gesamten Lebenszyklus auswirken können, um effiziente, qualitativ hochwertige und vor allem leicht zugängliche Behandlungen für alle Menschen weltweit zu entwickeln.
Der Lipidstoffwechsel und insbesondere das Cholesteringleichgewicht werden durch die HCV-Infektion beeinflusst. Die Korrelation zwischen Lipidstoffwechsel und HCV wurde klinisch seit langem beobachtet. In den Leberbiopsien von mit HCV infizierten Patienten wurde ein Anstieg der in den Lipidtröpfchen im Cytosol akkumulierten neutralen Lipide festgestellt (Dienes et al. 1982). Das Hepatitis-C-Virus wurde auch von Hypobetalipoproteinämie, Hypocholesterinämie und Lebersteatose begleitet (Schaefer und Chung 2013). Die Leber ist der primäre Ort für die Synthese, Speicherung und Oxidation von Lipiden und anderen Makromolekülen. Daher ist der Fettstoffwechsel in der Leber für die Aufrechterhaltung der systemischen Nährstoffhomöostase von wesentlicher Bedeutung. Eine Dysregulation des Leberlipidstoffwechsels ist ein Kennzeichen mehrerer Krankheiten wie Diabetes, alkoholische und nichtalkoholische Fettlebererkrankungen sowie parasitäre und virale Infektionen, einschließlich einer HCV-Infektion. (Erklärung der National Institutes of Health Consensus Development Conference: Management von Hepatitis C: 2002 - 10.-12. Juni 2002 2002; Fon Tacer und Rozman 2011; Chen et al. 2013; Reddy und Rao 2006; Visser et al. 2013; Wu und Parhofer 2014)
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The overall survival for patients with acute lymphoblastic leukemia (ALL) often is the function of age, in particular in 2019 analysis revealed that 5-year overall survival for patients older than 20 years remains below 35% (American Cancer Society, Cancer Facts &Figures 2019). Importantly, one of the major issues in ALL therapy is the ability of tumor cells to escape the treatment via the establishment of an immunosuppressive environment. The tumor microenvironment has gained tremendous importance in the past decade. This is largely based on the reasoning that, in order to devise better therapeutic strategies for patients, we need to gain better understanding into how malignant cells transform their microenvironment to promote growth, escape immune control and gain therapeutic resistance.
TAM receptors (TAMRs) are engaged in innate immune cells as a feed-back mechanism to terminate the immune response and promote the return to homeostasis (Rothlin et al. 2007). In the context of cancers, aberrant TAMR signaling was mainly explored concerning its pro-oncogenic function (Paolino and Penninger 2016). There are only limited data available suggesting the modulation of cancer immune response via TAMR signaling in highly immunogenic solid tumor models (Paolino et al. 2014; Ubil et al. 2018). So far, however, little is known about their potential indirect immune-modulatory function in hematological malignancies. Taking into account the pronounced importance of TAMR signaling in immune cells combined with the leukemic immune tolerance, the current study focused on the function of TAMR and their ligands in anti-leukemic immunity.
This work uncovers the mechanism of dampening anti-leukemic immune response via TAMR signaling on macrophages using the syngeneic BCR-ABL1 B-ALL mouse model. Using genetic depletion of GAS6 in the host environment or ablation of AXL and/or MERTK receptors in macrophages the bone marrow microenvironment could be rewired in order to achieve an efficient anti-leukemic immune response. In particular, the GAS6/AXL blockade triggers an effective NKand T- cell-dependent anti-leukemic response that results in prolonged survival. This finding specifically tackles the obstacle of inefficient bridging between innate and adaptive immune response typical for hematological malignancies in contrast to solid tumors (E. K. Curran, Godfrey, and Kline 2017).
Besides establishing the vital function of TAMR signaling in anti-leukemic immunity using murine models, the analysis of human blood plasma revealed that age-related immune dysregulation was manifested by significant GAS6 decrease and PROS1 upregulation among elderly donors (>60 y.o.) compared to controls (<25 y.o.). These data are indicative that TAMR signaling likely favors the age-dependent immune system decline, which in turn is associated with a poor survival rate of elderly patients diagnosed with leukemia.
In conclusion, using a preclinical ALL model here it was identified in vivo, that Axl significantly increases upon B-ALL challenge in Mph and NK cells. Therefore, AXL targeting, using the orally bioavailable selective inhibitor Bemcentinib, could serve as a powerful approach to revert early immunosuppression created by leukemia.
Taken together these data propose the AXL receptor as a novel immune checkpoint and attractive candidate for the development of a new therapeutic approach via unleashing the patient’s own immune system to combat leukemic cells.
At the beginning of the 1980s, an increased frequency of immune deficiency was discovered in a population of homosexual men, which is nowadays known as the Acquired Immune Deficiency Syndrome (AIDS). A few years later, the retro virus Human Immunodeficiency Virus 1(HIV-1) has been discovered as the cause of AIDS. Since the beginning of the pandemic, more that 74 million people have become infected and more than 32 million people died. In 2018, it was estimated that 38 million people where living with HIV-1 of which 24.5 million had access to Highly Active Antiretroviral Therapy (HAART), which blocks viral replication and prevents the progression towards AIDS. In the most cases an HIV-1 infection leads to the patient’s death within a few years Without HAART.
Taken together, this thesis shows that hematopoietic stem and progenitor cells harbor the prerequisites and characteristics to form an HIV-1 reservoir in vivo. The subsets of HSCs, MPPs and CD34+CD38+ progenitors harbor CD4 & CXCR4 double-positive cells as well as a lower amount of CD4 & CCR5 doublepositive cells. In addition, the susceptibility to X4-tropic HIV-1 is shown in vitro. Susceptibility to R5-tropic HIV-1 is only seen to a very low amount for CD34+CD38+ progenitors. The results also show that transduced HSPCs are capable to pass on integrated viral genomes via proliferation and differentiation during in vitro colony formation. More over the experiments provide evidence that this can take place for long time span as the outcome of the replating assays shows. Ex vivo analysis of HSPCs isolated from PLHIV also suggests that these cells are susceptible to HIV-1. Proviral DNA detection using a nested PCR showed infection of Lin- cells of a single donor with an R5-tropic subtype B HIV-1 clone. However, the assay could not detect infection of CD34+ cells. The
received results of this thesis are in agreement with previously published results. Albeit the obvious susceptibility to HIV-1 and existing reports of viral survival within HSPCs for several years, the low frequency of detected in vivo infected HSPCs could be related to the cytopathic effects of HIV-1 during replication resulting in cell death of potentially infected CD34+ cells. Other reasons could be associated with assay sensitivity or the small number of available patient samples. This makes hematopoietic stem and progenitor cells a target, which can be infected by HIV-1. The role and the clinical relevance of hematopoietic stem and progenitor cells in contribution to the latent viral HIV-1 reservoir within an HIV-1 infected patient needs to be further analyzed.
In the past decade, tissue-resident innate lymphoid cells (ILC) have become a central field of immunological research. ILC are a family of innate immune cells comprising cytotoxic Natural Killer (NK) cells and the non-cytotoxic helper like ILC1, ILC2 and ILC3. They mirror the functions and phenotypes of T cells, but do not require rearranged antigen-specific receptors for their rapid response to signals from injured or infected tissue. As potent cytokine producers being enriched in mucosal tissue, ILC play an essential role in tissue maintenance and regulating immunity to chronic inflammation and infection (Vivier et al., 2018). Although heterogeneity and plasticity of ILC complicates their classification, the pathophysiology of a broad variety of autoimmune and chronic inflammatory diseases have been associated with dysregulations in ILC subset distribution and functions (Dzopalic et al., 2019). This highlights their importance in human health and disease and accounts for the need for markers unambiguously describing the different ILC subtypes. This work introduces NKp65, a C-type lectin-like receptor (CTLR) encoded in the natural killer gene complex by the KLRF2 gene, as an exclusive marker for human ILC3. NKp65 expression especially discerns ILC3-like NK cell precursor from mature NK cells which express the NKp65-relative NKp80. Moreover, flow cytometric analysis of NKp65 expression aids in the demarcation of natural cytotoxicity receptor (NCR) expressing ILC3, from the closely related but functionally distinct RORt+ LTi cells and NCR- ILC3. This work further provides insights into NK cell development by in vitro differentiation studies in which NKp65 expressing cells are generated in presence of OP9 feeder cells and cytokines to support development. In such cultures, NKp65 expressing in vitro ILC (ivILC) acquire NKp80 expression in a Notch-dependent manner indicating their differentiation into mature NK cells. Acquisition of NK cell phenotypic markers is accompanied by NKp65 downregulation which leads to the mutually exclusive expression of NKp80 on NK cells and NKp65 on ILC3-like cells. Further insights are provided into the functional consequences of NKp65 engagement by its cognate high affinity ligand ‘keratinocyte-associated C-type lectin’ (KACL) which is selectively expressed on human keratinocytes (Bauer et al., 2015; Spreu et al., 2010). Expressed on ivILC, NKp65 mediates killing of KACL expressing target cells, suggesting that NKp65-KACL interaction promotes cellular cytotoxicity. In this context, the observed metalloproteinase dependent shedding of NKp65 might play a role in the termination of the cellular interaction. The findings on the regulation of NKp65 expression demonstrate the presence of a functional STAT5 response element in the KLRF2 promoter endowing a transcriptional control of NKp65 expression by IL-7 signaling. This provides an interesting link between the dependency of ILC3 on IL-7 signaling for their maintenance and the specific expression of NKp65 on these cells.
In summary, this study provides new insights into the physiologic expression of the CTLR NKp65 on human ILC3. The dependency of NKp65 surface expression on sustained STAT5 signaling provided by IL-7 underlines the connection of NKp65 expression and an ILC3 phenotype which might contribute to promote future research in discerning the interspersed pathways of ILC3 and NK cell development. The tissue and cell specific expression of NKp65 on ILC3 and its ligand KACL on keratinocytes of the human skin further suggests an important role of this genetically coupled receptor-ligand pair in tissue specific immunosurveillance.
The innate immune system is the first line of host defense that senses invading pathogens by various surveillance mechanisms, involving pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Furthermore, in response to stress, tissue injury or ischemia, cells release endogenous danger-associated molecular patterns (DAMPs) which activate PRRs in order to prompt an effective immune response. Activation of PRRs by DAMPs initiates signaling transduction pathways which drive sterile inflammation by the production of pro-inflammatory effector molecules. Biglycan, a class I small leucine-rich proteoglycan (SLRP), is proteolytically released from the extracellular matrix (ECM) in response to tissue stress and injury or de novo synthesized by activated macrophages. In its soluble form, biglycan operates as an ECM-derived DAMP and triggers a potent inflammatory response by engaging TLR2 and TLR4 on immune cells. By selective utilization of TLR2/4 and the TLR adaptor molecules adaptor molecule myeloid differentiation primary response gene 88 (MyD88) or TIR domain-containing adaptor-inducing interferon-β (TRIF) biglycan differentially regulates the production of TLR downstream mediators or inflammatory molecules. In this way, biglycan triggers the activation of mitogen-activated protein kinase (MAPK) p38, extracellular signal-regulated kinase (Erk) and nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in a primarily MyD88-dependent manner. In contrast, biglycan induces the expression of (C–C motif) ligand (CCL)5 and chemokine (C-X-C motif) ligand (CXCL)10 over TLR4/TRIF, heat shock protein 70 (HSP70) production over TLR2 and the synthesis of tumor necrosis factor (TNF)-α, CCL2 and CCL20 by utilizing TLR2/4/MyD88. As a consequence, biglycan promotes the recruitment of immune cells such as neutrophils, T cells, B cells and macrophages into the inflamed tissue. Research over the past years showed that biglycan-induced inflammation is involved in the pathogenesis of various inflammatory diseases such as lupus nephritis (LN), sepsis and renal ischemia/reperfusion injury (IRI), whereby genetic deletion of biglycan or TLR2/4 alleviated disease outcome. Unfortunately, the selective interaction of biglycan to TLRs and TLR adaptors complicates the identification of an efficient pharmacological target in biglycan-mediated inflammation. Yet, the necessity of possible co-receptors in biglycan signaling such as cluster of differentiation 14 (CD14) which was found in a high molecular complex with biglycan was not addressed so far.
In the first part of the present study, by utilizing primary peritoneal murine macrophages we demonstrated that the biglycan-induced expression and synthesis of TNF-α and CCL2 via TLR2/4/MyD88, CCL5 through TLR4/TRIF and HSP70 over TLR2 is blunted in CD14 deficient mice, proving that CD14 is essential in TLR2- and TLR4-mediated biglycan signaling. Pre-incubation of macrophages with an anti-CD14 antibody significantly reduced the protein levels of TNF-α, CCL2, CCL5 and HSP70. In line with these data, pharmacological inhibition of CD14 alleviated the transcriptional activation of NF-κB by biglycan in HEK-Blue cells expressing hTLR2/CD14 as well as hTLR4/CD14/MD2 supporting CD14-dependency for biglycan/TLR2/4 signaling. Western blot analysis of phosphorylated p38, p44/42 and NF-κB in WT and CD14 deficient mice revealed that activation of biglycan-mediated TLR downstream signaling is CD14-dependent. Accordingly, biglycan-induced activation and nuclear translocation of p38, p44/42 and NF-κB was blocked in Cd14-/- mice as analyzed by confocal microscopy. Co-immunoprecipitation studies combined with microscale thermophoresis analysis showed that biglycan is in complex with CD14 in macrophages and in vitro binds directly with high affinity to CD14, thereby sustaining the concept that CD14 is a novel co-receptor in biglycan-mediated inflammation. Additionally, we provided proof-of-principle of our concept in an in vivo mouse model of renal IRI. Transient overexpression of biglycan in WT mice exacerbated the expression and production of TNF-α, CCL2, CCL5 and HSP70 in a CD14-dependent manner. Interestingly, pLIVE or pLIVE-hBGN-injected Cd14-/- mice displayed lower chemo- and cytokine levels in reperfused kidneys as compared to respective WT controls during renal IRI (30 h), indicating a renoprotective effect by CD14 deficiency. Flow cytometry analysis of kidney homogenates underlined the pivotal effect of CD14 in biglycan signaling as biglycan-mediated infiltration of CD11b- and F4/80-positive renal macrophages was abolished in Cd14-/- mice. Additionally, pLIVE or pLIVE-hBGN-injected CD14 deficient mice displayed lower numbers of renal CD11b- and F4/80-positive cells during renal IRI compared to WT mice. Analysis of F4/80- and CD38-positive cells isolated from mononuclear cell extracts from kidney homogenates of pLIVE or pLIVE-hBGN-injected WT and Cd14-/- mice revealed that biglycan triggers the polarization of pro-inflammatory M1 macrophages in a CD14-dependent manner. In line with this, Cd14-/- mice, either injected with pLIVE or pLIVE-hBGN, showed less F4/80- and CD38-positive cells during renal IRI than the respective WT control. As a corroboration of our data PAS-stained renal sections of pLIVE- or pLIVE-hBGN-injected WT or Cd14-/- mice uncovered that biglycan worsens tubular damage in IRI-subjected mice via CD14. At the same time, tubular damage was significantly reduced in IRI-subjected Cd14-/- mice as compared to WT mice. In correlation with these data, serum creatine levels were increased in pLIVE-hBGN-injected WT mice during renal IRI. In contrast, serum creatine levels were significantly less increased in pLIVE- or pLIVE-hBGN-injected Cd14-/- mice than in WT littermate controls. In conclusion we demonstrated that CD14 is a new high affinity ligand for biglycan-mediated pro-inflammatory signaling over TLR2 and TLR4 in macrophages. In vivo, soluble biglycan triggers the expression of various inflammatory mediators by utilizing the co-receptor CD14. Ablation of CD14 abolishes biglycan-induced renal macrophage infiltration and M1 macrophage polarization as well as overall kidney function by reduced tubular damage and serum creatinine levels. Therefore, this study identifies CD14 as a promising therapeutic target to ameliorate biglycan-induced inflammation.
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Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas in Western Europe. The nodular sclerosing subtype of cHL (NS cHL) is characterised by a proliferation of fibroblasts in the tumour microenvironment, leading to fibrotic bands surrounding the lymphoma infiltrate. Several studies have described a crosstalk between the tumour cells of cHL, the Hodgkin- and Reed-Sternberg (HRS) cells, and cancerassociated fibroblasts (CAF). However, to date a deep molecular understanding of these fibroblasts is lacking. Aim of the present study therefore was a comprehensive
characterisation of these fibroblasts. Moreover, only a few studies describe the interplay of HRS cells and CAF. The paracrine communication and direct interaction of these two
cellular fractions have been investigated within this study. Finally, the influence of a few HRS cells within a lymph node orchestrate the mere alteration of its architecture and
morphology. Gene expression and methylation profiles of fibroblasts isolated from primary lymph node suspensions revealed persistent differences between fibroblasts obtained from NS cHL and lymphadenitis. NS cHL derived fibroblasts exhibit a myofibroblastic - inflammatory phenotype characterised by MYOCD, CNN1 and IL-6 expression. TIMP3, an inhibitor of matrix metalloproteinases, was strongly upregulated in NS cHL fibroblasts, likely contributing to the accumulation of collagen in sclerotic bands of NS cHL. Treatment by luteolin could reverse this fibroblast phenotype and decrease TIMP3 secretion. NS cHL fibroblasts showed enhanced proliferation when they were exposed to soluble factors released from HRS cells. For HRS cells, soluble
factors from fibroblasts were not sufficient to protect them from Brentuximab-Vedotin(BV) induced cell death. However, HRS cells adherent to fibroblasts were protected from BV-induced injury. The cHL specific interaction of both cell fractions reveals an initiation of inflammatory key regulators such as IL13 and IL4. Among important adhesion molecules known from literature the blocking of integrin beta 1 solely interrupted the adhesion of HRS cells to CAF. In summary, this study proves the stable reprograming of CAF phenotype and expression derived from NS cHL. It presents a suitable in vitro model for studying the interaction of HRS cells and CAF by paracrine factors and adherence. Most importantly the observations confirm the importance of fibroblasts for HRS cells´ inflammatory niche and cell survival associated with TIMP3 which probably acts as a major factor to the typical accumulation of fibrosis observed in NS cHL.