RNU rats have been used to prove the biocompatibility of the matrix grafts. This should be a surrogate for human patients mimicking an allogenic implantation setting. RNU rats are an athymic specific breed of rats which are deficient in T-cells. The acute reaction and rejection of foreign materials should be suppressed. Although the immune reaction is compromised, the model has still a preserved inflammatory response to foreign material . To examine the graft-host compatibility, decellularized grafts have been implanted subcutaneously into the rats. At graft retrieval after 7 days, no macroscopic foreign body reaction was evident. Histological examination showed cell migration of host cells into both grafts. Early cell infiltration with macrophages was shown to be a good predictor of further graft behavior. It has been demonstrated that a lower M1/M2 ratio and increased M2 macrophage infiltration at early time points are beneficial for graft remodeling . However, tissue treated with cross-linking agents and decellularizing detergents can negatively disturb the M1/M2 macrophagepolarization, leading to graft failure . Therefore, to distinguish specific cells responsible for inflammation, rejection, and remodeling, cell subsets were characterized by using specific antibodies. The immunohistochemistry showed that both grafts were infiltrated with macrophages, T-cells, and B-cells. As expected, a low infiltration of T-cells occurred due to the immunodeficient animal model. The grafts retrieved after 7 days were mostly infiltrated with macrophages. Comparing the samples for pan macrophages (CD68) and M2 macrophages (CD163), it could be assumed that M2, was more dominant than the M1 macrophages. This could explain the progressive graft degradation . Further analysis of the biocompatibility showed that both grafts did not reveal a significant difference in cell infiltration. For a significantly higher cell migration into the scaffolds, a 7-day observation period might not be long enough. However, the histological samples of grafts retrieved at day 28 could not provide any more detail. As a result of progressive degeneration, differentiation between subcutaneous tissue and graft was not possible. Therefore, further cell migration could not be quantified.
A series of intracellular molecules including signal transducer and activator of transcription (STAT) fam- ily members, peroxisome proliferator-activated recep- tor γ (PPARγ), interferon regulatory factors (IRFs), and microRNAs were identified as key modulators of microglia/macrophagepolarization [ 17 ]. Interestingly, using a model of apoptosis in human neuroblastoma cells, Ulbrich and colleagues observed that argon af- fected the phosphorylation and binding activity of STAT3, and that inhibition of STAT3 attenuated ar- gon’s anti-apoptotic effect [ 25 ]. It is reasonable to as- sume that argon influences the activation and function of STAT3, which in turn produces an effect on microglia/macrophagepolarization, and ultimately leads to behavioral and histological improvements after ischemic stroke. However, further studies will be needed to confirm this hypothesis. Moreover, some signaling pathways, such as mammalian target of rapamycin complex 1 (mTORC1) pathway and 5′ AMP-activated protein kinase (AMPK) pathway, were also identified recently to be essential for microglia/ macrophagepolarization after stroke [ 52 , 53 ]. The roles of these molecules and pathways in the regula- tion of microglia/macrophagepolarization and the neuroprotection induced by argon treatment should be further investigated.
were respectively enhanced, in LMP2 -/- or LMP7 -/- cells, compared to wt cells. Among these genes, 4 well described M2 marker genes (Arg1, Retnla, Ccl17 and Mcr1) were selected for the further qPCR validation for their expression, which confirmed the enhancement of M2 marker gene expression and thus M2 polarization in LMP2-/- and LMP7 -/- AMs. Most notably, the IL-4 dependent Th2 chemokine CCL17 was released in high amounts from LMP7 and LMP2 deficient AMs. Our findings that LMP2 and -7 similarly affect M2 polarization were further corroborated by analysis of early signaling kinetics upon IL-4 stimulation. In particular, expression of the M2 specific transcription factor Irf4 - a key transcription factor that controls M2 macrophagepolarization (80) was disproportionately higher in M2 polarized LMP2 -/- and LMP7 -/- cells and its expression exceeded wt levels particularly in the first 48 h after IL-4 induction. Similar results were obtained for STAT6 and AKT activation, all hallmarks of IL-4 mediated signaling towards M2 polarization (83, 188): Both STAT6 and AKT phosphorylation were enhanced in LMP2 -/- and LMP7 -/- cells, respectively. In addition, we are the first to find that IRF4 expression was induced in M2 but suppressed in M1 AMs, which fits well the literature. Previous studies have proven that IRF4 is the downstream target of STAT6 activation, and in addition, there is a protein-protein interaction between IRF4 and STAT6. Hence, we believe that the IRF4 service is a positive loop to cooperate with STAT6 to derive M2 markers gene expression.
5.2.6 Mutation of the PS binding site in SphK2 prevents its cleavage
As noticed in Figure 16C I observed a residual activity of caspase-1 in Jurkat cells, which was also evident in most other cell lines that were used. Therefore, additional regulatory mechanisms, besides caspase-1 activation, must be critical in regulating SphK2 release during apoptosis. Interestingly, SphK2 as well as SphK1 contain phosphatidylserine (PS) binding sites, which are well conserved between both isoenzymes (Figure 19). Indeed, PS was shown to stimulate the activity of both isoforms (117). PS exposure on the outer leaflet of AC is considered to be a hallmark of apoptosis and crucial for provoking anti-inflammatory macrophagepolarization after their interaction with AC (160). I therefore questioned whether PS exposure would affect the release or activity of SphK2 during apoptosis. The critical amino acid residues for PS binding to SphK1 have been identified and were shown to have a profound impact on SphK1 membrane targeting (161). I used this knowledge and mutated the analogous threonine 219 to alanine in SphK2.
Secreted LCN2 forms complexes with bacterial siderophores (Goetz et al, 2002), which can be internalized via two putative receptors, the 24p3 receptor (Slc22a17, BOCT) and megalin (Chakraborty et al, 2012; Devireddy et al, 2005) (see Figure 9). Recently, Devireddy and colleagues reported the existence of mammalian siderophores (Devireddy et al, 2010). Depending on the iron status of the LCN2- siderophore complexes (iron loaded (holo-LCN2) or iron-lacking (apo-LCN2)) it was proposed that the internalization process leads to either iron transport or apoptosis, respectively. Uptake of apo-LCN2 was shown to lead to the induction of the proapoptotic protein BCL2L11, a mitochondrial protein of the Bcl2 family (Devireddy et al, 2005). Associated with this concept, Liu et al. found increased cell numbers in the bone marrow, the peripheral blood and the spleen in LCN2-deficient mice when compared to wild-type littermates (Liu et al, 2011). The authors argued that these differences resulted from the defective apoptosis of neutrophils, thymocytes, erythroid cells and cytokine-dependent mast cells (Liu et al, 2011). Another group assessed the effects of LCN2 on human hematopoiesis and found that LCN2 induced apoptosis in mature cells of the erythroid and monocyte/macrophage lineage (Miharada et al, 2008). However, in another report, LCN2 was found to protect immune cells from apoptosis during endotoxic shock in mice (Srinivasan et al, 2012a).
On a basal level, M2(IL10) and TAM-like macrophages showed the lowest OCR in Seahorse measurements, while M2(IL4) macrophages had the highest consumption of oxygen (Figure 7 A,E). A similar pattern was observed for the subsets in terms of spare respiratory capacity (SRC, Figure 7 G), considered as an indicator of how efficiently cells can adapt to changing energy demands [ 17 ]. The extracellular acidification rate as an indirect measurement of glycolysis revealed the highest basal levels in M1 and M2(IL4) macrophages (Figure 7 C,F). ThioA injection reduced OXPHOS-dependent ATP production (Figure 7 B) and caused a compensatory ECAR increase (Figure 7 D). In comparison to oligomycin, these effects were achieved more slowly, leading to higher minimal OCR values after injection (Figure 7 H). Due to donor-specific differences in the bioenergetic profile of in vitro polarized macrophage subsets, individual graphs are shown in Figure S9.
Pro-inflammatory M1, also termed “classically activated macrophages” are polarized by stimulation of T helper (T H ) 1 cytokines including interferon (IFN) γ or by pattern- associated molecular patterns, such as lipopolysaccharide (LPS). M1 macrophages produce cytokines like tumor necrosis factor (TNF) α, interleukin (IL)-6, IL1β, IL-12 and IL-23 and express major histocompatibility complex (MHC)-II, CD80, CD86 and CD68. M1 cells produce chemotactic proteins to attract T H 1 cells e.g., C-X-C motif ligand (CXCL) 9 and CXCL10. Intracellular proteins also involved in polarization processes are interferon-regulatory factors (IRF), suppressor of cytokine signaling (SOCS) and signal transducers and activators of transcription (STAT). IRF1, IRF5 and IRF8 were demonstrated to induce M1 macrophages (Gunthner & Anders, 2013). Activation of Stat1 and Socs1 in mice are important for M1 polarization (Murray, Allen et al., 2014), as well as myeloid differentiation primary response gene 88 (MyD88) dependent pathway. Ligation of toll-like receptor (TLR) ligands, of which TLR4 and TLR2 are highly expressed, and subsequent activation of nuclear factor kappa B (NFκB), phosphoinositid-3-kinase (PI3K) and IFNγ secretion, induces inducible nitric oxide synthase( iNOS) in M1 macrophages (Castoldi et al., 2015).
An essential feature of macrophages is their high plasticity, which allows them to adopt diverse phenotypes in response to equally diverse microenvironmental conditions. Moreover, plasticity is required due to the broad functional spectrum of macrophages ranging from inflammation, host defense, tissue remodeling, and even metabolism (Biswas et al., 2012; Geeraerts et al., 2017). In case of bacterial infection or injury, tissue-resident macrophages and monocytes, recruited from the blood and differentiated into macrophages, induce a protective inflammatory response. It can be divided into different phases, which merge fluently into each other: from pathogen distruction and removal of cellular debris to repairing tissues repair and maintainance of homeostasis (Italiani and Boraschi, 2014; Sica et al., 2015; Ginhoux et al., 2016; Atri, Guerfali and Laouini, 2018). These phases are accompanied by different macrophage activation states induced by the respective cues, such as microbial components, cytokines, or fatty acids. This phenomenon of versatile phenotype adoption is termed “macrophagepolarization” and is a com- plex spatiotemporal process (Mantovani et al., 2004; Sica et al., 2015; Atri, Guerfali and Laouini, 2018; Shapouri-Moghaddam et al., 2018). Based on cell surface markers, production of specific factors, and biological activities, several subtypes of macrophages have been described in mice and humans (Shapouri-Moghaddam et al., 2018). For the purpose of simplification, however, two major polarization programs have been suggested, mirroring the Th1/Th2 polarization scheme of T helper cells: classically activated macrophages or M1 and alternatively activated macrophages or M2, schematically depicted in figure 1-1 (Mantovani et al., 2002, 2004; Biswas and Mantovani, 2012; Sica et al., 2015; Murray, 2017). Originally, the concept of macrophagepolarization was defined in vitro using transcriptional profiling and conventional approaches. However, such polarization states can also be observed under physiological (ontogenesis, preg- nancy) and pathological (allergic and chronic inflammation, cancer) conditions in vivo (Sica et
Sphingosine-1-phosphate (S1P) is an important membrane derived, pleotropic signaling lipid that plays prominent roles in several diseases [16–19]. Especially in the context of inflammation, this lipid been shown to regulate various cellular events such as chemotaxis, apoptosis, phagocytosis, and macrophagepolarization [19,20]. Intracellularly, S1P levels are dynamically regulated at picomolar concentrations by its synthesis and degradation by various enzymes, including sphingosine kinase (SPHKs), S1P lyases (SGPLs), and S1P phosphatases (SGPPs). S1P is generated from sphingosine by phosphorylation, which is carried out by two metabolically redundant kinases, i.e., SPHK1 and SPHK2. S1P binds to its specific G-protein-coupled receptors (S1PR1–S1PR5) to elicit cell responses. Besides, it can also bind to intracellular targets such as histone deacetylases and TNFR-associated factor 2 (TRAF2) [21,22]. Intracellular S1P can also act as a second messenger to trigger calcium release from the endoplasmic reticulum [23–25]. In spite of demarcated receptor-dependent and independent actions of S1P, the autocrine and paracrine functions of S1P in innate immune cells such as macrophages are still elusive, especially in the context of NLRP3 inflammasome activation. Furthermore, factors upstream to S1PRs were not systematically investigated as a putative therapeutic targets in inflammation and cancer. In this study, we sought to characterize autocrine and paracrine functions of S1P in macrophages and to define components of S1P production or its signaling machinery that is best suited to target S1P-dependent NLRP3 inflammasome activation and IL-1β production.
which were not in the scope of NMR until now. NMR signal enhancement is achieved by transferring large electron polarization to surrounding nuclei via DNP. In the last decade, there has been remarkable progress in solution and solid-state DNP-NMR as well as in imaging through the application of hyperpolarization methods. 4–7 As pointed out recently, this ‘‘renaissance of DNP’’ combined with high magnetic fields will further grow in the coming years, enabling demanding applications with better resolution. 8,9 Materials and biological systems such as membrane or micro- crystalline proteins, 10–13 fibrils, 14 ligands bound to receptors, 15 polymers, 16–18 surface-bound species, 19–23 and other low concen- trated samples were studied beneficially using DNP-NMR. 13,24–26 Cryogenic temperatures are required to slow down nuclear and electron relaxation and to allow an efficient polarization transfer in solid-state DNP NMR experiments. In contrast to biological systems, 15,27–29 most technically relevant materials do not show detrimental lines broadening at low temperatures.
The term job polarization was popularized by Goos, Manning (2007). They build upon the hypothesis of Autor, Levy, Murnane (2003) that technological change is routine- biased, in order words that technological change is complementary to interactive tasks at the upper tail of the wage distribution and erodes demand for routine tasks in the middle, but is neutral to non-routine unskilled tasks, such as those in personal services. When occupations are ranked according to their initial average wage, jobs at both ends grow more strongly than those in the middle of the distribution. The result is the U- shaped wage/employment profile familiar from many recent studies.
In order to assess the functional role of MIF in acute wound healing disorders, we first focused on its ability to mobilize PBMCs to the site of inflammation. The ability of MIF to pro- mote leukocyte recruitment was described earlier [ 30 ]. But in contrast to employing exoge- nously added cytokines, we used supernatants from whole adipose tissue that secrete a mixture of soluble factors containing stimulatory and inhibitory effects. MCP-1 is a well-known chemo- kine and served as a positive control [ 20 ]. We showed that PBMC chemotaxis was partially abrogated by MIF antibodies. Furthermore, our results indicate that MIF and MCP-1 may have an additive effect on PBMC chemotaxis. The chemotactic effect was not completely neu- tralized by the combined administration of anti-MIF and anti-MCP-1 antibodies implying that other soluble factors such as fatty acids may mediate the chemotaxis of inflammatory cells into adipose tissue [ 20 , 31 ]. To underscore our in vitro observations, we investigated the accumula- tion of labeled macrophages into LPS-injected epididymal fat pads of WT and Mif –/– mice. LPS was used as a stimulus to produce local adipose tissue inflammation [ 32 , 33 ]. Mif –/– mice showed a significantly lower infiltration rate of FITC-labeled macrophages when compared to WT mice which confirms the potential importance of MIF in cell recruitment to acutely inflamed adipose tissue. Our findings are in line with Verschuren et al. who observed decreased macrophage infiltration in white adipose tissue in Mif-deficient mice with lower expression of intercellular adhesion molecule (I-CAM)-1 and CD44, two relevant adhesion molecules induc- ing monocyte infiltration [ 34 ]. Although our immunohistochemical staining showed a higher ATM infiltration in IAT, an inflammatory-dependent increase of MIF expression in adipocytes or ATMs could not be demonstrated. Future studies are required to evaluate the source of MIF secretion in adipose tissue.
RANKL and M-CSF induced osteoclastogenesis of resident BM and ES macrophages
Osteoclasts develop in the bone matrix, where they secrete acid and lytic enzymes for the re- sorption of bone, and maintain mineral homeostasis. Osteoclastogenesis proceeds from mono- cytes/macrophages through an osteoclast precursor, a prefusion osteoclast, and a fused polykaryon to a mature osteoclast at the bone interface. This process is mainly mediated by the receptor activator of NF-kappa B ligand (RANKL) and the macrophage colony stimulat- ing factor (M-CSF) (Boyle et al. 2003; Zaidi 2007). M-CSF induces proliferation of the os- teoclast precursors, whereas RANKL induces the switch to the prefusion osteoclast, indicated by the onset of tartrate resistant acid phosphatase (TRAP) expression and activity (Troen 2004). Sørensen and colleagues showed that osteoclasts can be derived from peripheral blood monocytes (Sorensen et al. 2007). Expression analysis on the mRNA and protein level showed that both macrophage subtypes expressed the necessary receptors, RANK and CD115. TRAP activity measurements revealed no TRAP activity in unstimulated BM and ES macrophages, and induced TRAP activity after M-CSF and RANKL treatment in both macro- phage subtypes. The fusion of osteoclast precursor cells to polykaryons is necessary for the development of mature osteoclasts (Boyle et al. 2003). Both macrophage subtypes formed cell clusters, but fusion to polykaryons was not observed. Thus, for both BM and ES macrophages M-CSF and RANKL treatment resulted in the induction of osteoclastogenesis, resting in the prefusion osteoclast stage.
Palmitate treatment of monocytes leads to macrophage inflammatory protein 1-alpha and beta upregulation (MIP- 1α/β, also known as CCL3 and 4, respectively) and this occurs in a MAPK, NF-κB, and PI3K dependent manner indicating that PI3Ks can directly promote FFA mediated inflammation ( 69 , 70 ). Interestingly, both chemokines are involved in neutrophil and monocyte recruitment, respectively ( 71 ), suggesting FFA mediated PI3K dependent signaling could promote increases in ATM number. Further evidence that palmitate mediated PI3K activation within myeloid cells regulates ATM content is provided by observations that palmitate treatment of macrophages induces netrin-1 and its receptor Unc5b, mediators that promote ATM retention and accumulation ( 72 ). Interestingly, in other cellular systems, netrin-1 acts in concert with its receptor in a PI3K dependent manner ( 73 ), although the functional relevance of PI3Ks to palmitate mediated ATM retention remains unexplored. These studies suggest that PI3Ks integrate signals derived from FFAs and thereby influences ATM accumulation and inflammatory status. However, many of the studies cited are limited by their exclusive use of in vitro models, disregarding the complexity of signals present in vivo.
Derzeit wird nach neuen Therapieansätzen zur effektiven medikamentösen Bekämpfung der Legionellose und der Melioidose gesucht. Hierbei spielt der bereits erwähnte Virulenzfaktor „macrophage infectivity potentiator“-Protein, welches im nachfolgenden als Mip bezeichnet wird, eine entscheidende Rolle. Sowohl das Mip von Legionella pneumophila (LpMip) als auch das Mip von Burkholderia pseudomallei (BpMip) zählen zur Familie der FK506-Bindungsproteine (FKBPs). Die FKBPs wiederum lassen sich in die Superfamilie der Peptidyl-Prolyl-cis/trans-Isomerasen (PPIasen) einordnen, welche die Fähigkeit besitzen, die cis/trans-Isomerisierung von Peptidbindungen der Aminosäure Prolin zu katalysieren. 48 Da bei dieser Aminosäure - im Gegensatz zu anderen - der Betrag der Freien Energie der beiden Konformeren geringer ist, gibt es keine energetische Präferenz für eine der beiden Konformationsisomeren. Deshalb kann die Sekunden oder Minuten dauernde Umwandlung des bei ca. 5 - 6 % vorliegenden cis-Isomers in das trans-Isomer beobachtet werden. 49
Der Prozess der Entzündung und die Entstehung von Neoplasien sind durch komplexe Abläufe innerhalb des Organismus gekennzeichnet, die einer angemessenen Kontrolle des Zellzyklus und der Proliferation bedürfen. Dabei spielt eine Reihe von Eiweißstof- fen, sowohl im Entzündungsgeschehen als auch in der Entstehung von Malignomen, eine wesentliche Rolle. Das proinflammatorische Zytokin Macrophage Migration Inhibitory Factor (MIF) ist eines dieser Proteine, welches einen bedeutenden Beitrag in der Proliferation und Differenzierung von Zellen leistet. MIF stimuliert die Tumorzell- proliferation, die Migration und die Metastasierung, fördert die Tumorangiogenese und hemmt die p53-vermittelte Apoptose. Aufgrund einer vermehrten Expression von MIF in diversen malignen Tumoren interessierte das Vorkommen von MIF im Zervixkarzinom. In der vorliegenden Arbeit wurde der Expressionsstatus von MIF im Zervixkarzinom auf mRNA- und Proteinebene mittels Western Blot und Immunhistochemie untersucht.
2009). Ideological scores are available for two-year congress terms. We take absolute values of these scores to measure political polarization. The unit of our analysis is an incumbent representative who is reelected at least once.
To measure the incumbent’s performance, we build two indicators for the voters’ assess- ment of their representatives’ work on natural disaster relief. Both indicators are based on the data from the Federal Emergency Management Agency. First, we construct an aggregate measure of post-disaster treatment duration for the incumbent’s state during his previous term in office. We expect a negative effect such that a shorter post-disaster treatment makes the voters perceive the incumbents as more competent and so leads to higher political po- larization. Our second measure of the incumbents’ performance in natural disaster relief is a dummy for public assistance programs initiated after the disaster declarations. For this measure, we expect a positive effect on political polarization because a public assistance pro- gram initiated after the disaster makes the voters perceive their representatives as being more competent.