Sebastian Temme

The Herpes Simplex Virus-1 Encoded Glycoprotein B Diverts HLA-DR into the Exosome Pathway

Neutralizing Abs play an important role for immunity against HSV-1 infection. This branch of the immune response is initiated by MHC class II Ag presentation and activation of T cell help. In this study, we show that the HSV-1 encoded glycoprotein B (gB) manipulates the class II processing pathway by perturbing endosomal sorting and trafficking of HLA-DR (DR) molecules. Expression of gB in the human melanoma cell line Mel JuSo results in formation of enlarged DR+ intracellular vesicles. Costaining of the vesicles revealed the presence of DR, gB, and the late endosomal marker CD63. The lumen of these late endosomal membranes shows a variable content, containing either gB or CD63, or both CD63 and gB. gB targets DR molecules on their biosynthetic route, after the MHC class II invariant chain is released from the DR heterodimer. gB-DR complexes were detected in a post-Golgi compartment and in exosomes, but not on the cell surface. Interestingly, increasing expression of gB strongly elevated the amount of DR and CD63 released into the exosome pathway. In conclusion, this is a previously undescribed mode of viral immune evasion involving hijacking of DR from its normal transport route to the cell surface, followed by viral-mediated release of DR into the exosome pathway.

Selective Activation of Adenosine A2A Receptors on Immune Cells by a CD73-Dependent Prodrug Suppresses Joint Inflammation in Experimental Rheumatoid Arthritis

Phosphorylated adenosine A2A receptor agonists suppressed inflammation in a model of arthritis without A2A-mediated vasodilatory side effects. Separating the Wheat from the Chaff Extolling the virtues of simple building design, the modern architect Ludwig Mies van der Rohe famously declared that “less is more,” a philosophy that applies to modern drug design as well. Because simpler drugs have fewer side effects, the promise of adenosine A2A receptor agonists as therapeutics would grow if one could only separate their anti-inflammatory and vasodilator functions. Now, Flögel et al. built an adenosine A2A receptor agonist (chet-AMP) that displays only the anti-inflammatory function in an animal model of rheumatoid arthritis. How the authors accomplished this feat lies “in the details,” to again paraphrase van der Rohe. To isolate the anti-inflammatory effects of A2AR, Flögel et al. synthesized a prodrug that required, for its activation, the presence of ecto-5′-nucleotidase (CD73), which is mainly found on endothelial and immune cells. Using 19F magnetic resonance imaging to track inflammation noninvasively over time, the authors showed that chet-AMP, but not chet-adenosine, reduced inflammation in a mouse model of collagen-induced arthritis. This effect was dependent on the presence of both CD73 and A2AR, and no vasodilation was observed until drug concentrations were increased 100-fold. Physicians most often use corticosteroids to treat inflammatory conditions, but these drugs, although effective, can cause serious complications. By simplifying delivery of a drug only to the sites where it is needed most, Flögel et al. quell inflammation and avoid an undesirable side effect. Adenosine A2A receptor (A2AR) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A2AR agonists (prodrugs) that require the presence of ecto-5′-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5′-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 (19F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A2AR. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A2AR expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A2AR agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation.

Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half-lives and sensitivity.

Inflammatory processes can reliably be assessed by 19F MRI using perfluorocarbons (PFCs), which is primarily based on the efficient uptake of emulsified PFCs by circulating cells of the monocyte–macrophage system and subsequent infiltration of the 19F‐labeled cells into affected tissue. An ideal candidate for the sensitive detection of fluorine‐loaded cells is the biochemically inert perfluoro‐15‐crown‐5 ether (PFCE), as it contains 20 magnetically equivalent 19F atoms. However, the biological half‐life of PFCE in the liver and spleen is extremely long, and so this substance is not suitable for future clinical applications. In the present study, we investigated alternative, nontoxic PFCs with predicted short biological half‐lives and high fluorine content: perfluorooctyl bromide (PFOB), perfluorodecalin (PFD) and trans‐bis‐perfluorobutyl ethylene (F‐44E). Despite the complex spectra of these compounds, we obtained artifact‐free images using sine‐squared acquisition‐weighted three‐dimensional chemical shift imaging and dedicated reconstruction accomplished with in‐house‐developed software. The signal‐to‐noise ratio of the images was maximized using a Nutall window with only moderate localization error. Using this approach, the retention times of the different PFCs in murine liver and spleen were determined at 9.4 T. The biological half‐lives were estimated to be 9 days (PFD), 12 days (PFOB) and 28 days (F‐44E), compared with more than 250 days for PFCE. In vivo sensitivity for inflammation imaging was assessed using an ear clip injury model. The alternative PFCs PFOB and F‐44E provided 37% and 43%, respectively, of the PFCE intensities, whereas PFD did not show any signal in the ear model. Thus, for in vivo monitoring of inflammatory processes, PFOB emerges as the most promising candidate for possible future translation of 19F MR inflammation imaging to human applications. Copyright

19F magnetic resonance imaging of endogenous macrophages in inflammation.

In this article, we review the use of (19) F MRI (magnetic resonance imaging) for in vivo tracking of monocytes and macrophages in the course of tissue inflammation. Emulsified perfluorocarbons (PFCs) are preferentially phagocytized by monocytes/macrophages and are readily detected by (19) F MRI. Because of the lack of any (19) F background in the body, observed signals are robust and exhibit an excellent degree of specificity. As a consequence of progressive infiltration of the labeled immunocompetent cells into inflamed areas, foci of inflammation can be localized as hot spots by simultaneous acquisition of morphologically matched proton ((1) H) and fluorine ((19) F) MRI. The identification of inflammation by (19) F MRI--at a time when the inflammatory cascade is initiated--opens the possibility for an early detection and more timely therapeutic intervention. Since signal intensity in the (19) F images reflects the severity of inflammation, this approach is also suitable to monitor the efficacy of pharmaceutical treatment. Because PFCs are biochemically inert and the fluorine nucleus exhibits high magnetic resonance (MR) sensitivity, (19) F MRI may be applicable for clinical inflammation imaging.

Ecto-5′-Nucleotidase on Immune Cells Protects From Adverse Cardiac Remodeling

Rationale: Ecto-5′-nucleotidase (CD73) on immune cells is emerging as a critical pathway and therapeutic target in cardiovascular and autoimmune disorders. Objective: Here, we investigated the role of CD73 in postinfarction inflammation, cardiac repair, and remodeling in mice after reperfused myocardial infarction (50-minute ischemia). Methods and Results: We found that compared with control mice (1) cardiac function in CD73−/− mice more severely declined after infarction (systolic failure with enhanced myocardial edema formation) as determined by MRI and was associated with the persistence of cardiac immune cell subsets, (2) cardiac adenosine release was augmented 7 days after ischemia/reperfusion in control mice but reduced by 90% in CD73 mutants, (3) impaired healing involves M1-driven immune response with increased tumor necrosis factor-&agr; and interleukin-17, as well as decreased transforming growth factor-&bgr; and interleukin-10, and (4) CD73−/− mice displayed infarct expansion accompanied by an immature replacement scar and diffuse ventricular fibrosis. Studies on mice after bone marrow transplantation revealed that CD73 present on immune cells is a major determinant promoting cardiac healing. Conclusions: These results, together with the upregulation of CD73 on immune cells after ischemia/reperfusion, demonstrate the crucial role of purinergic signaling during cardiac healing and provide groundwork for novel anti-inflammatory strategies in treating adverse cardiac remodeling.

CD73 on Immune Cells Protects from Adverse Cardiac Remodeling

Rationale: Ecto-5′-nucleotidase (CD73) on immune cells is emerging as a critical pathway and therapeutic target in cardiovascular and autoimmune disorders. Objective: Here, we investigated the role of CD73 in postinfarction inflammation, cardiac repair, and remodeling in mice after reperfused myocardial infarction (50-minute ischemia). Methods and Results: We found that compared with control mice (1) cardiac function in CD73−/− mice more severely declined after infarction (systolic failure with enhanced myocardial edema formation) as determined by MRI and was associated with the persistence of cardiac immune cell subsets, (2) cardiac adenosine release was augmented 7 days after ischemia/reperfusion in control mice but reduced by 90% in CD73 mutants, (3) impaired healing involves M1-driven immune response with increased tumor necrosis factor-&agr; and interleukin-17, as well as decreased transforming growth factor-&bgr; and interleukin-10, and (4) CD73−/− mice displayed infarct expansion accompanied by an immature replacement scar and diffuse ventricular fibrosis. Studies on mice after bone marrow transplantation revealed that CD73 present on immune cells is a major determinant promoting cardiac healing. Conclusions: These results, together with the upregulation of CD73 on immune cells after ischemia/reperfusion, demonstrate the crucial role of purinergic signaling during cardiac healing and provide groundwork for novel anti-inflammatory strategies in treating adverse cardiac remodeling.

Noninvasive Imaging of Early Venous Thrombosis by 19F Magnetic Resonance Imaging With Targeted Perfluorocarbon Nanoemulsions

Background— Noninvasive detection of deep venous thrombi and subsequent pulmonary thromboembolism is a serious medical challenge, since both incidences are difficult to identify by conventional ultrasound techniques. Methods and Results— Here, we report a novel technique for the sensitive and specific identification of developing thrombi using background-free 19F magnetic resonance imaging, together with &agr;2-antiplasmin peptide (&agr;2AP)–targeted perfluorocarbon nanoemulsions (PFCs) as contrast agent, which is cross-linked to fibrin by active factor XIII. Ligand functionality was ensured by mild coupling conditions using the sterol-based postinsertion technique. Developing thrombi with a diameter <0.8 mm could be visualized unequivocally in the murine inferior vena cava as hot spots in vivo by simultaneous acquisition of anatomic matching 1H and 19F magnetic resonance images at 9.4 T with both excellent signal-to-noise and contrast-to-noise ratios (71±22 and 17±5, respectively). Furthermore, &agr;2AP-PFCs could be successfully applied for the diagnosis of experimentally induced pulmonary thromboembolism. In line with the reported half-life of factor XIIIa, application of &agr;2AP-PFCs >60 minutes after thrombus induction no longer resulted in detectable 19F magnetic resonance imaging signals. Corresponding results were obtained in ex vivo generated human clots. Thus, &agr;2AP-PFCs can visualize freshly developed thrombi that might still be susceptible to pharmacological intervention. Conclusions— Our results demonstrate that 1H/19F magnetic resonance imaging, together with &agr;2AP-PFCs, is a sensitive, noninvasive technique for the diagnosis of acute deep venous thrombi and pulmonary thromboemboli. Furthermore, ligand coupling by the sterol-based postinsertion technique represents a unique platform for the specific targeting of PFCs for in vivo 19F magnetic resonance imaging.

γ-Interferon-regulated chaperone governs human lymphocyte antigen class II expression

Antigen presentation by human lymphocyte antigen (HLA) class II peptide receptors alerts the immune system to infections. In antigen‐presenting cells (APCs), HLA class II, HLA‐DM, and associated invariant chain‐encoding genes are exclusively regulated by the interferon γ (IFNγ)‐inducible class II transactivator (CIITA). Control of CIITA expression could therefore govern expression of class II peptide receptors in the diverse group of APCs. We discovered that elevation of the HLA class III region encoded B‐associated transcript 3 (BAT3) increases and depletion of BAT3 decreases expression of HLA class II, HLA‐DM, and invariant chain. IFNγ strongly elevates BAT3 transcription in various tumor cell lines and in primary macrophages. BAT3 chaperones the simultaneously IFNγ‐induced CIITA. Following IFNγ‐treatment, both CIITA and BAT3 translocate from the cytosol to the nucleus. The nuclear import of CIITA mediated by IFNγ controls activation of HLA class II genes. BAT3 is a novel key regulator of components of the HLA class II processing pathway. We present a mechanism explaining how parallel IFNγ‐mediated regulation of CIITA and of its chaperone BAT3 controls the level of components of the HLA class II processing pathway.—Kämper, N., Franken, S., Temme, S., Koch, S., Bieber, T., Koch, K. γ‐Interferon‐regulated chaperone governs human lymphocyte antigen class II expression. FASEB J. 26, 104–116 (2012). www.fasebj.org

Stoichiometry of HLA class II-invariant chain oligomers.

Background The HLA gene complex encodes three class II isotypes, DR, DQ, and DP. HLA class II molecules are peptide receptors that present antigens for recognition by T lymphocytes. In antigen presenting cells, the assembly of matched α and β subunits to heterodimers is chaperoned by invariant chain (Ii). Ii forms a homotrimer with three binding sites for class II heterodimers. The current model of class II and Ii structure states that three αβ heterodimers bind to an Ii trimer. Methology/Principal Findings We have now analyzed the composition and size of the complexes of class II and Ii using epitope tagged class II subunits and density gradient experiments. We show here that class II-Ii oligomers consist of one class II heterodimer associated with one Ii trimer, such that the DR, DQ and DP isotypes are contained within separate complexes with Ii. Conclusion/Significance We propose a structural model of the class II-Ii oligomer and speculate that the pentameric class II-Ii complex is bent towards the cell membrane, inhibiting the binding of additional class II heterodimers to Ii.

Technical Advance: Monitoring the trafficking of neutrophil granulocytes and monocytes during the course of tissue inflammation by noninvasive 19F MRI

Inflammation results in the recruitment of neutrophils and monocytes, which is crucial for the healing process. In the present study, we used 19F MRI to monitor in vivo the infiltration of neutrophils and monocytes from the onset of inflammation to the resolution and healing phase. Matrigel, with or without LPS, was s.c.‐implanted into C57BL/6 mice. This resulted in a focal inflammation lasting over a period of 20 days, with constantly decreasing LPS levels in doped matrigel plugs. After i.v. administration of 19F containing contrast agent, 19F MRI revealed a zonular 19F signal in the periphery of LPS containing matrigel plugs, which was not observed in control plugs. Analysis of the 19F signal over the observation period demonstrated the strongest 19F signal after 24 h, which decreased to nearly zero after 20 days. The 19F signal was mirrored by the amount of leukocytes in the matrigel, with neutrophils dominating at early time‐points and macrophages at later time‐points. Both populations were shown to take up the 19F contrast agent. In conclusion, 19F MRI, in combination with the matrigel/LPS model, permits the noninvasive analysis of neutrophil and monocyte infiltration over the complete course of inflammation in vivo.