Host Genetic Background Strongly Influences the Response to Influenza A Virus Infections

The genetic make-up of the host has a major influence on its response to combat pathogens. For influenza A virus, several single gene mutations have been described which contribute to survival, the immune response and clearance of the pathogen by the host organism. Here, we have studied the influence of the genetic background to influenza A H1N1 (PR8) and H7N7 (SC35M) viruses. The seven inbred laboratory strains of mice analyzed exhibited different weight loss kinetics and survival rates after infection with PR8. Two strains in particular, DBA/2J and A/J, showed very high susceptibility to viral infections compared to all other strains. The LD50 to the influenza virus PR8 in DBA/2J mice was more than 1000-fold lower than in C57BL/6J mice. High susceptibility in DBA/2J mice was also observed after infection with influenza strain SC35M. In addition, infected DBA/2J mice showed a higher viral load in their lungs, elevated expression of cytokines and chemokines, and a more severe and extended lung pathology compared to infected C57BL/6J mice. These findings indicate a major contribution of the genetic background of the host to influenza A virus infections. The overall response in highly susceptible DBA/2J mice resembled the pathology described for infections with the highly virulent influenza H1N1-1918 and newly emerged H5N1 viruses

Cloning and expression of eq.IFNg, eq.GM-CSF and eq.IL-4 and their influence on monocytic horse cells

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Problemstellung 3\. Material+Methoden1 4\. Material+Methoden2 5\. Material+Methoden3 6\. Ergebnisse 7\. Diskussion 8\. Zusammenfassung 9\. Summary Literaturverzeichnis</aIn der vorliegenden Arbeit sollten dendritische Zellen (DC) aus der Haut von Pferden isoliert und aus Monozyten differenziert und charakterisiert werden. Zu diesem Zweck war es zunächst notwendig, die eq.Zytokine, Granulozyten- Makrophagen Kolonie-stimulierender Faktor (GM-CSF), Interleukin 4 (IL-4) und Interferon g (IFN g) herzustellen. Eq.GM-CSF wurde zunächst mit Hilfe von Consensusprimern, die aus anderen Spezies abgeleitet wurden, amplifiziert und hergestellt. Nach Durchführung des RACE-Verfahrens konnte eine Punktmutation (Deletion) im Bereich des 3´Primers erkannt werden, die dazu führte, daß die Proteinsequenz 8 Aminosäuren länger ist als bei anderen bekannten Spezies. Bei eq.IL-4 stellte sich heraus, daß die veröffentlichten Sequenzinformationen nicht korrekt waren, da sie Deletionen enthielten. Lediglich die Sequenzinformationen zu eq.IFNg konnten bis auf eine Punktmutation bestätigt werden. Um aktive rekombinante Proteine herzustellen, wurden pro- und eukaryotische Expressionssysteme verwendet. Im bakteriellen System konnte für alle Zytokine eine Überexpression gezeigt werden. Sie zeigten jedoch aufgrund des gewählten Expressionssystemes keine biologische Aktivität. In mammalia Zellen exprimierte Zytokine waren bioaktiv. Langerhans Zellen (LC) konnten aus der Epidermis isoliert und morphologisch untersucht werden. Die Ausbeute war jedoch sehr gering. Die beim Pferd isolierten peripheren Blutmonozyten (PBM) wurden mit rekombinanten Zytokinen differenziert. Diese monozytären Zellen wurden morphologisch und funktionell charakterisiert. Mit eq.IFNg stimulierte Monozyten zeigten Charakteristika von Makrophagen (große adhärente Zellen mit hochregulierter MHC II Expression). In licht- und elektronenmikroskopischen Untersuchungen sowie Analysen der Oberflächenproteine im Durchflußzytometer zeigten die mit eq.GM-CSF und eq.IL-4 stimulierten Monozyten Ähnlichkeit zu den humanen MoDC (Schleierzellen mit Ausläufern mit hochregulierter MHC II und CD86 Expression). Tests in der gemischten Leukozytenreaktion zeigten, daß die MoDC Stimulationskapazität für T Zellen besaßen.In this thesis, dendritic cells (DC) should be isolated from the skin of horses. Additionally monocytes should be differentiated to DC and characterised. For this purpose, it was necessary to produce the equine (eq.) cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4) and interferon g (IFNg). With the help of consensus primers from other species, eq.GM-CSF was amplified and produced. After the RACE-procedure, a point mutation (deletion) along a section of the 3´primer, which resultated in a protein sequence 8 amino acids longer than other known species, was identified. The published sequence information of eq.IL-4 was revealed to be incorrect because it contained deletions. Only the sequence information for eq.IFNg -except for one point mutation- could be confirmed. To produce active recombinant proteins, both prokaryotic and eukaryotic expression systems were used. In the bacterial system an over expression of all cytokines was possible but, due to the system chosen, had no biological activity. The expressed cytokines from mammalia cells were bioactive. Langerhans cells (LC) isolated from the epidermis were examined morphologically, however the cell yield was very low. The isolated peripheral blood monocytes (PBM) from horses were differentiated with the recombinant cytokines. These monocytic cells were characterised morphologically as well as functionally. Eq. IFNg stimulated monocytes showed characteristics of macrophages (large adherent cells with highly regulated MHC II expression). Using light- and electron- microscopic thechniques as well as analysis of the surface proteins by flow cytometry, there was found similarity between the eq .GM-CSF and the eq.IL-4 stimulated monocytes with human MoDC (veiled cells with pseudopodia and with highly regulated MHC II/CD86 expression). Tests of the mixed leukocyte reaction showed that the MoDC possessed stimulation capacity for T cells

Virulence Determinants of Avian H5N1 Influenza A Virus in Mammalian and Avian Hosts: Role of the C-Terminal ESEV Motif in the Viral NS1 Protein ▿

We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain- and host-dependent manner

MAP1S controls breast cancer cell TLR5 signaling pathway and promotes TLR5 signaling-based tumor suppression.

Targeting TLR5 signaling in breast cancer represents a novel strategy in cancer immunotherapy. However, the underlying mechanism by which TLR5 signaling inhibits cancer cell proliferation and tumor growth has not been elucidated. In this study, we found TLR5 agonist flagellin inhibited the cell state of activation and induced autophagy, and reported that autophagy protein MAP1S regulated the flagellin/TLR5 signaling pathway in breast cancer cells through enhancement of NF-κB activity and cytokine secretion. Remarkably, MAP1S played a critical role in tumor suppression induced by flagellin, and knockdown of MAP1S almost completely abrogated the suppression of tumor growth and migration by flagellin treatment. In addition, elevated expression of MAP1S in response to flagellin feed-back regulated tumor inflammatory microenvironment in the late stages of TLR5 signaling through degradation of MyD88 in autophagy process. These results indicate a mechanism of antitumor activity that involves MAP1S-controlled TLR5 signaling in breast cancer

DBA/2J mice are highly susceptible to H7N7 (SC35M) virus infection compared to C57BL/6J mice.

<p>(A): DBA/2J and C57BL/6J mice were infected intra-nasally with 2×10³ FFU SC35M virus. DBA/2J (B) and C57BL/6J (C) mice were infected with increasing doses of SC35M (H7N7) virus via the intra-nasal route. Weight loss and survival of infected mice was followed over a period of 14 days. Mortality includes also mice that were sacrificed because they had lost more than 25% of body weight. Data are from two independent experiments. Mean percent of body weight change (±SEM) is shown. DBA/2J and C57BL/6J groups were compared for statistically significant differences using non-parametric Mann-Whitney-U-test. *: p value<0.05; **: p<0.01; ***: p value<0.001.</p

DBA/2J mice are highly susceptible to PR8 infections compared to C57BL/6J mice.

<p>DBA/2J (A) and C57BL/6J (B) mice were infected with increasing doses of PR8 virus via the intranasal route and survival was recorded for the following 14 days. Mortality includes also mice that were sacrificed because they had lost more than 25% of body weight.</p

Male and female mice of DBA/2J and C57BL/6J mice show similar weight loss and survival after infection with Influenza A virus.

<p>DBA/2J female, DBA/2J male, C57BL/6J female and C57BL/J male were infected intra-nasally with 2×10³ FFU PR8 virus. Weight loss and survival of infected mice was followed over a period of 14 days. Mortality includes also mice that were sacrificed because they had lost more than 25% of body weight. Mean percent of body weight change (±SEM) is shown. p-values for significance were calculated for pair wise comparison between all groups and for all days using non-parametric Mann-Whitney-U-test. On days 2–4, all DBA/2J male and female groups differed significantly in their weight loss from the C57BL/6J groups (p<0.001 for all comparisons). No consistently significant difference was observed between male and female C57BL/6J groups (except at day 4, p<0.05), whereas the male and female DBA/2J groups were significantly different at days 2–4 (p<0.05 at day 2 and p<0.01 at days 3, 4).</p

DBA/2J mice exhibit a stronger inflammatory response than C57BL/6J mice.

<p>DBA/2J (checked bars) and C57BL/6J mice (black bars) were infected intra-nasally with 2×10³ FFU of PR8 virus. Bronchio-alveolar lavage (BAL) was collected from non-infected controls (c) or at the indicated days (d1, d2, d3, d4) post infection, and the concentration of cytokines (A) or chemokines (B) was determined. Expression of cytokines and chemokines was determined by real-time PCR (C). Each time point represents the mean value ±SEM of 7 mice per group for (A) and (B), and of 10 mice per group for (C). DBA/2J and C57BL/6J mice were compared for statistically significant differences using non-parametric Mann-Whitney-U-test. *: p value<0.05; **: p<0.01; ***: p value<0.001.</p