The Tyrosine Kinase Btk Regulates the Macrophage Response to <i>Listeria monocytogenes</i> Infection

<div><p>In this study we investigated the role of Bruton's tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium <i>Listeria monocytogenes</i> (<i>Lm</i>). In response to <i>Lm</i> infection, Btk was activated in bone marrow-derived macrophages (BMMs) and <i>Btk</i>^−/− BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of <i>E. coli</i> fragments, there was no difference between wt and <i>Btk</i>^−/− BMMs in the uptake of <i>Lm</i> upon infection. Moreover, there was no difference in the response to heat-killed <i>Lm</i> between wt and <i>Btk</i>^−/− BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular <i>Lm</i>. Finally, <i>Btk</i>^−/− mice displayed enhanced resistance and an increased mean survival time upon <i>Lm</i> infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in <i>Btk</i>^−/− mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during <i>Lm</i> infection.</p> </div

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model

<div><p>Background</p><p>Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated <i>in-vitro</i> and <i>in-vivo</i>.</p><p>Principal Findings</p><p>We show <i>in-vitro</i> that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection.</p><p>Conclusion</p><p>A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.</p></div

Normal cytokine production in response to hk<i>Lm</i> and Pam₃CSK₄.

<p>(A) Semi-quantivative RT-PCR analysis showing the expression of <i>Tlr2</i> in wt and <i>Btk</i>^{−<i>/</i>−} BMMs. <i>Hprt</i> was used as an input control. Data are representative of two independent experiments. (B) Wt and <i>Btk</i>^{−<i>/</i>−} BMMs were stimulated (+) with hk<i>Lm</i> (10⁸ cells/ml) or with Pam₃CSK₄ (1 µg/ml), or were left non-treated as a control (−) for 24 hours. Afterwards, the cytokine levels in the supernatants were determined by ELISA. Data show the summary of 4–13 independent experiments (hk<i>Lm</i>: TNF-α, n = 4, IL-6, n = 5; Pam₃CSK₄: TNF-α, n = 8, IL-6, n = 9). For each experiment, wt cytokine levels were set as 1 and the relative levels in supernatants from <i>Btk</i>^{−<i>/</i>−} BMMs were calculated. Mean with SEM is shown. The cytokine levels in individual batches of wt BMMs for hk<i>Lm</i> were in the range of 2.3–9.6 ng/ml for TNF-α and 0.9–13.4 ng/ml for IL-6. For Pam₃CSK₄the range was 0.6–19.1 ng/ml for TNF-α and 2.3–39.5 ng/ml for IL-6.</p

Btk-deficient mice show reduced susceptibility to <i>Lm</i> infection.

<p>(A) Left panel: Kaplan-Meier plot showing the survival of wt and <i>Btk</i>^{−<i>/</i>−} mice after <i>Lm</i> (EGD, 10⁶ CFU) infection over a period of 14 days. Right panel: Diagram indicates the mean survival time of wt and <i>Btk</i>^{−<i>/</i>−} mice after <i>Lm</i> infection. Data show the summary of two independent experiments with a total of 23 wt and 24 <i>Btk</i>^{−<i>/</i>−} mice. (B) Wt and <i>Btk</i>^{−<i>/</i>−} mice were infected with <i>Lm</i> (EGD, 10⁶ CFU) and serum IFN-γ (left) and IL-12p70 (right) levels were measured on day 1, 2 and 3. n = 5 (day 1), 6 (day 2), 5 (day 3) for IFN-γ and 5 (day 1), 5 (day 2), 5 (day 3) for IL-12p70. (C) Wt and <i>Btk</i>^{−<i>/</i>−} mice were infected with <i>Lm</i> (EGD, 10⁶ CFU) and CFU-spleen and CFU-liver were measured on day 5. n = 5 (wt) and 8 (Btk-null). (A–C) Mean with SEM is shown. Survival data were analyzed using a log rank (Mantel-Cox) test, while the other P-values were calculated using an unpaired Student's t-test. *, P≤0.05; **, P≤0.01; n.s. not significant.</p

Btk is activated upon <i>Lm</i> infection.

<p>(A) Diagrams showing the total BMM cell number (left; n = 8) and the percentage of F4/80⁺CD11b^hi (right; n = 2) BMMs at day 9 of culture. Mean with SEM is shown. (B) RT-PCR analysis showing the expression of <i>Bmx</i>, <i>Btk</i>, <i>Itk</i>, <i>Rlk</i> and <i>Tec</i> in wt and <i>Btk</i>^{−<i>/</i>−} BMMs. <i>Hprt</i> was used as a loading control. To confirm the functionality of the primers used for RT-PCR, the expression of <i>Btk</i>, <i>Itk</i>, <i>Rlk</i> and <i>Tec</i> in BM-derived mast cells (BMMC) and of <i>Bmx</i> in BM is shown. Data are representative of two independent experiments. (C) Immunoblot analysis showing the activation of Btk in wt BMMs. Cells were infected with <i>Lm</i> (LO28, MOI 10) for the indicated time periods and the autophosphorylation of Y223 was monitored in whole cell lysates. Total Btk levels serve as a loading control. Data are representative of four independent experiments. (D) Summary of the quantification of all immunoblots analyzed (n = 4). The relative intensity was calculated as the ratio of pY223-Btk/total Btk intensity for each time-point. Subsequently, the ratio in non-infected wt BMMs (time-point 0 min) was set as one and the relative levels for the other time points were calculated. The band intensity was evaluated using MultiGauge software. The P-value was calculated using an unpaired Student's t-test.</p

Analysis of the phagocytic function of <i>Btk</i>^{−<i>/</i>−} BMMs and uptake of <i>Lm.</i>

<p>(A) Wt and <i>Btk</i>^{−<i>/</i>−} BMMs were incubated with fluorescein-labeled <i>E. coli</i> particles for two hours and the fluorescence of phagocytosed particles was measured. Diagram indicates the fluorescence intensity by phagocytosed <i>E. coli</i> particles. Data show the summary of three independent experiments with a total of four independent cell batches. (B) Wt and <i>Btk</i>^{−<i>/</i>−} BMMs were infected with CFSE-labeled <i>Lm</i> (LO28, MOI 40) and three hours later the cells were fixed, permeabilized and cellular actin was stained with Phalloidin-Alexa. The total number of intracellular bacteria and the number of cytoplasmic bacteria co-localizing with host actin were determined by fluorescence microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060476#s2" target="_blank">material and methods</a>. The diagram on the left indicates the mean number of <i>Lm</i> per cell. The right diagram displays the percentage of <i>Lm</i> that escaped to the cytoplasm. For each experiment, the number of <i>Lm</i> in 50 infected wt or <i>Btk</i>^{−<i>/</i>−} BMMs was counted. Data are representative of two independent experiments. (A and B) Mean with SEM is shown. The P-values were calculated using an unpaired Student's t-test. ***, P≤0.001; n.s. not significant.</p

Isobologram of compound interaction.

<p>Comparison of the combination of different doses of both compounds necessary to reach 50% replication inhibition of (A) H7N7 and (B) H1N1(09)pdm (◆) to a model of dose additivity that would represent a curve progression of 1 (□). Dose response was tested with an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. On the x-axis the concentration of Zanamivir and on the y-axis the concentration of carrageenan is presented. The concentrations (determined as mean of 3 replicates) are given as the fraction of the respective IC₅₀ values of the different viruses with the particular compound (IC₅₀ = 1).</p

Therapeutic efficacy in influenza H1N1(09)pdm lethally infected mice.

<p>Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan and Zanamivir (1 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant.</p

IC₅₀ values of carrageenan and Zanamivir for influenza A viruses of human and animal origin.

<p>^a IC₅₀ values were calculated in comparison to untreated infected cells. Each value represents the mean IC₅₀ of 6 replicates/assay and their standard deviation.</p><p>IC₅₀ values of carrageenan and Zanamivir for influenza A viruses of human and animal origin.</p