The role of B cells during acute and latent infections with murine cytomegalovirus and Leishmania major

This thesis focuses on the role of B cells in mCMV and Leishmania major infection. B cells are an essential component of the adaptive immune system and play a key role in the humoral immune response. In mCMV infection we analyzed the influence of B cells on the virus-specific CD8 T cell response, in detail the role of B cells as IL-10 secreting cells, as source of immunoglobulin (Ig) and as antigen presenting cells. In Leishmania major infection we investigated the role of Ig in Th1 and Th2 directed disease.rnWe found in mCMV infection that the B cell secreted IL-10 suppresses effectively the acute virus-specific CD8 T cell response, while the IL-10 secreted by dendritic cell has no obvious effect. Ig has no effect in the acute virus-specific CD8 T cell response, but in memory response Ig is essential. If Ig is missing the CD8 T cell population remains high in memory response 135 days post infection. The complete absence of B cells dramatically reduces the acute virus-specific CD8 T cell response, while B cell reconstitution just partially rescues this dramatic reduction. A comparison of this reduction in a B cell free organism to an organism with depleted dendritic cells gave a similar result. To exclude a malfunction of the CD8 T cells in the B cell deficient mice, the decreased virus-specific CD8 T cell population was confirmed in a B cell depletion model. Further, bone marrow chimeras with a B cell compartment deficient for CD40-/- showed a decrease of the virus-specific response and an involvement of CD40 on B cells. Taken together these results suggest a role for B cells in antigen presentation during mCMV infection.rnFurther we took advantage of the altered mCMV specific CD8 T cell memory response in mice without Ig to investigate the memory inflation of CD8 T cells specific for distinct mCMV specifc peptides. Using a SIINFEKL-presenting virus system, we were able to shorten the time until the memory inflation occurs and show that direct presentation stimulates the memory inflation. rnIn Leishmania major infection, Ig of Th2 balanced BALB/c mice has a central role in preventing a systemic infection, although the ear lesions are smaller in IgMi mice without specific Ig. Here the parasite loads of ears and spleen are elevated, and an IMS-reconstitution does not affect the parasite load. In contrast in Th1 balanced C57BL/6 mice, reconstitution of IgMi mice with serum of either untreated or immunized mice decreased the parasite load of spleen and ear, further IMS treatment reduces the size of the spleen and the cytokine-levels of IL-10, IL-4, IL-2 and IFN-γ to a level comparable to wt mice. rnIn der vorliegenden Arbeit wird die Rolle von B Zellen in mCMV und in Leishmania major Infektion im Mausmodell untersucht. B Zellen sind eine essentielle Komponente des adaptiven Immunsystems und übernehmen eine Schlüsselrolle in der humoralen Immunantwort. In der mCMV Infektion wurde der Einfluß von B Zellen auf die virus-spezifische CD8 T Zellantwort analysiert. Dabei wurden unterschiedliche Aspekte der B Zellen, Einfluss auf die Immunantwort zu nehmen, wie die Sekretion von IL-10, als Antikörperquelle und als Antigen präsentierende Zellen, untersucht. Bei der Leishmania major-Infektion lag der Fokus auf der Untersuchung des Einflusses von Antikörpern auf die Th1 und die Th2 Antwort.rnIn mCMV Infektionen konnte gezeigt werden, dass von B Zellen sekretiertes IL-10 die virus-spezifische CD8 T Zellantwort supprimiert, im Gegensatz zu IL-10 sekretiert von dendritischen Zellen, das die virus-spezifische CD8 T Zellantwort nicht merklich beeinflusst. Die von B Zellen sekretierten Antikörper haben nur in der ‚memory’-Antwort das Gewicht die virus-spezifische CD8 T Zellantwort zu beeinflussen; fehlen Antikörper, verbleibt die virus-spezifische CD8 T Zellantwort 135 Tage nach Infektion auf dem Niveau der akuten Antwort. Das komplette Fehlen von B Zellen hingegen führt zu einer enormen Verringerung in der akuten CD8 T Zell Antwort, die durch Rekonstitution mit B Zellen nur partiell aufgehoben werden konnte. Ein Vergleich der Immunantwort in einem B Zell-defizienten Organismus mit einem dendritische Zellen depletierten Organismus zeigte dieselbe verminderte Immunantwort. Durch einen weiteren Vergleich eines B Zell defizienten Organismus mit einem B Zell depletierten Organismus konnte eine Beeinträchtigung der CD8 T Zellen in dem B Zell defizienten Organismus ausgeschlossen werden. Knochenmarkschimären mit einer B Zell Population ohne CD40 Expression zeigten, dass CD40 auf B Zellen einen Einfluss auf die virus-spezifische CD8 T Zellantwort hat, die in Abwesenheit von CD40 verringert ist. Diese Ergebnisse implizieren eine Rolle von B Zellen als Antigen präsentierende Zellen hin.rnDes Weiteren wurde die veränderte virus-spezifische CD8 T Zellantwort in Abwesenheit von Antikörpern benutzt, um ein Phänomen der ‚memory’-Antwort zu untersuchen, ‚memory inflation’ genannt. Die durchgeführten Untersuchungen führten zu einem experimentellen System, in dem die Zeitspanne bis zum erkennbaren Auftreten der ‚memory inflation’ verkürzt werden konnte. Es konnte auch nachgewiesen werden, dass die für die ‚ memory inflation’ charakteristische virus-spezifische CD8 T Zell Population durch direkte Präsentation stimuliert wird. rnIn Leishmania major Infektion hängt die Immunantwort vom genetischen Hintergrund der Mäuse ab. BALB/c Mäuse antworten mit einer Th2 Antwort, in der Antikörper eine zentrale Rolle bei der Prävention vor einer systemischen Infektion spielen. Während in C57BL/6 Mäuse der Leishmania major Infektion mit einer Th1 Antwort begegnet wurde: Durch die Rekonstituierung von Mäusen ohne Antikörpern mit Serum von bereits Leishmania major infizierten Mäusen oder unimmunisierten Mäuse konnte eine starke Verbesserung erzielt werden, resultierend waren die Parasitenlasten in der Milz und den Ohren nach der Rekonstituierung vergleichbar mit den gemessenen in Wildtyp Mäusen, das Gleiche galt für die Größe der Milz sowie den Zytokinen IL-10, IL-4, IL-2 und IFN-γ. r

Efficient B cell depletion via diphtheria toxin in CD19-Cre/iDTR mice

B cells were first discovered as antibody producing cells, as B-1 B cells and finally as effector cells. In recent years their capacity to serve as antigen presenting cells is increasingly appreciated, and better tools are needed to study their function. We have previously described a new mouse model, the iDTR mice, that allow for the Cre-mediated expression of the diphtheria toxin receptor, thus rendering cells that express the Cre-recombinase sensitivity to diphtheria toxin. Herein we describe a new mouse line, the B-DTR mice, where the CD19-Cre was crossed to the iDTR mice. B-DTR allows for the efficient and cost-effective depletion of different B cell subpopulations, but only partially plasma cells. These mice can therefore be used to study the importance of B cells versus plasma cells in different immune responses and autoimmune diseases

eYFP expression in CD19-Cre/eYFP mice.

<p>A. CD19-Cre/eYFP mice were analyzed for eYFP expression in the B cell compartment of spleen, lymph nodes, Peyer's patches, peritoneal cavity and bone marrow. The shown histograms are gated on live lymphocytes and CD19. The filled histogram curve represents CD19-Cre/eYFP mice and the unfilled control mice. Percentages of eYFP⁺ CD19⁺ cells are shown in the histograms, upper number/percentage B-DTR mice, lower panel shows cells of control mice. B. To identify the CD19⁺ eYFP⁻ B-cell fraction in bone marrow of CD19-Cre/eYFP mice, bone marrow cells were stained for AA4.1 and CD23.</p

Diphtheria toxin mediated depletion of B cells in B-DTR mice.

<p>The mice were treated once daily for 4 days with a dose of 25 ng/g body weight of diphtheria toxin. The decrease of B cells in comparison to T cells is shown in spleen, lymph nodes, bone marrow and peritoneal cavity; dot blots in the left panel and the corresponding bar charts in the right panel.</p

Plasma cells identified with Syndecan-1 in cytospins.

<p>Bone marrow cells from NP-CG immunized and boosted cells were analyzed for plasma cells. Plasma cells are shown in red with a surface staining of syndecan-1, the remaining cell population was counter stained with Hoechst (blue). The bar chart represents plasma cells found in the regarding groups (n = 5). The pictures show representative sections of the cytospins.</p

Efficiency of B cell depletion.

<p>A. Number of marginal zone, follicular, immature and mature B cells in spleen. B. Depletion of B cells in bone marrow, gated for B220⁺. C. Depletion of transitional B cells in spleen, gated for CD19⁺ cells. D. Depletion of mature and immature B cells in spleen, gated for CD19⁺ cells. E. Depletion of marginal zone and follicular B cells gated for CD19⁺ cells. F. Depletion of B1a and B2 B cells in peritoneal cavity, gated for CD19⁺. G. Depletion of resting B cells in peritoneal cavity gated for CD19⁺ the percentages of cells in the different shown B cell subpopulations are indicated.</p

Total serum immunoglobulin levels after depletion of B cells.

<p>B-DTR mice and control mice were injected daily four times with 25 ng/g body weight DT. Mice were bled just before DT injection and in weekly intervals thereafter. Shown are the total immunoglobulin levels at the indicated time points. The values are shown as average of groups of at least five mice.</p

Impact of a Single Dose of a Probiotic Nutritional Supplement (AB001) on Absorption of Ethylalcohol: Results From a Randomized Double-Blind Crossover Study

Background: We conducted a prospective placebo-controlled double-blind randomized Study to assess the impact of a single dose of a nutritional Supplement (AB001) on alcohol absorption in healthy subjects. Other objectives were the impact on breath alcohol content, cognitive function 1 hour after alcohol uptake and tolerability. Method: A total of 24 healthy volunteers were enrolled into the study (12 male, 12 female, age: 28.3 ± 10.8 years, BMI: 23.5 ± 5.7 kg/m²). On the experimental day, they ingested a light breakfast together with a single dose (2 capsules) of AB001 (or placebo) and drank 2 moderate glasses of spirit (a total of 0.6 g/kg body weight). Breath alcohol tests and blood draws for determination of blood alcohol levels were performed for up to 6 hours. After crossover, the experiment was repeated in the following week. Areas under the curves were calculated to determine alcohol absorption rates. Results: There was a significant reduction of blood alcohol by 10.1% ( P  < .001) with AB001, when compared to placebo. There was a less pronounced but also significant reduction of alcohol in the breath test by 7.2% ( P  < .05). No difference in the cognitive function test between AB001 and placebo could be observed 60 minutes after alcohol ingestion (22.6 ± 8.0 seconds vs 23.0 ± 11.2 seconds, n.s.). The supplement uptake was well tolerated and there were no adverse events related to the study intervention. Conclusion: Uptake of a single dose of AB001 shortly before drinking alcohol significantly reduced plasma alcohol and breath alcohol concentrations, but the effect was less pronounced compared to chronic uptake as shown previously