Immunological mechanisms of control of pneumococcal carriage.

<p>The serotype-dependent and -independent immunological mechanisms that control pneumococcal carriage are depicted. Memory B cells and plasma cells specific to capsule and proteins produce IgG. This can lead to antibody-mediated pneumococcal agglutination and antibody-mediated phagocytosis by neutrophils, monocytes, and macrophages. Moreover, IL-17A produced by memory CD4⁺ T cells might lead to recruitment and activation of neutrophils and monocytes/macrophages, thus increasing phagocytosis. CCL2, C-C motif chemokine ligand 2; CD4⁺, cluster of differentiation 4; IgG, immunoglobulin G; IL-17A, interleukin 17-A.</p

Intact Type I Interferon Production and IRF7 Function in Sooty Mangabeys

<div><p>In contrast to pathogenic HIV/SIV infections of humans and rhesus macaques (RMs), natural SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high viremia. Several studies suggested that low immune activation and relative resistance of CD4+ central memory T-cells from virus infection are mechanisms that protect SMs from AIDS. In 2008 it was reported that plasmacytoid dendritic cells (pDCs) of SMs exhibit attenuated interferon-alpha (IFN-α) responses to TLR7/9 ligands <i>in vitro</i>, and that species-specific amino acid substitutions in SM Interferon Regulatory Factor-7 (IRF7) are responsible for this observation. Based on these findings, these authors proposed that “muted” IFN-α responses are responsible for the benign nature of SIV infection in SMs. However, other studies indicated that acutely SIV-infected SMs show robust IFN-α responses and marked upregulation of Interferon Stimulated Genes (ISGs). To investigate this apparent disparity, we first examined the role of the reported IRF7 amino acid substitutions in SMs. To this end, we sequenced all IRF7 exons in 16 breeders, and exons displaying variability (exons 2,3,5,6,7,8) in the remainder of the colony (177 animals). We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles. In addition, functional assays using recombinant SM IRF7 showed no defect in its ability to translocate in the nucleus and drive transcription from an IFN-α promoter. Furthermore, <i>in vitro</i> stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV_mac239 induced an 500–800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans. These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.</p></div

Transactivation of IFNA4 promoter and nuclear localization by Sooty Mangabey IRF7.

<p>(A) HEK293 cells were transfected with the luciferase reporter plasmid containing the human IFNA4 promoter, TBK1 and IRF7 constructs from human, rhesus, and sooty mangabeys, or vehicle, as indicated. Luciferase expressed from the SV40 promoter was transfected as a positive control. Luciferase activity was measured 24 h after transfection. Values represent the average of triplicate wells for Rhesus and Sooty-IRF7 and duplicate wells for the remaining samples. Data are representative of three individual experiments. (B) COS-7 cells were transfected with Sooty-IRF7-GFP and TBK1, or with vehicle for 24 hrs, then stained with DAPI. Magnification is indicated to the right of panels. Data are representative of three experiments.</p

Sooty mangabey pDCs produce IFN-α in response to TLR7 agonists and SIVmac239.

<p>(A) PBMCs from SIV-negative SMs and SIV-negative RMs were incubated for 18 hr with 10 µM CL097 or 3 µg/ml SIVmac239 and stained for intracellular IFN-α. The lower panels depict uninfected human PBMCs stimulated with 10 µM CL097 or 3 µg/ml AT2 HIV-1. Insets denote percentages of IFN-α+ pDCs within the pDC population. (B) Cumulative data for CL097 stimulations, average percentage of pDCs expressing IFN-α are denoted by horizontal bars. (C) Percentage of IFN-α+ pDCs after 18 hr stimulation with AT2 SIV_mac239, or AT2 HIV-1 (for human PBMCs). Means are shown by horizontal bars. The species (RM, SM or HU) from which PBMCs were prepared and virus strain used for stimulation is depicted below the X-axis. (D) RNA production of IFN-α in PBMCs from SMs and RMs was assessed using qPCR. Fold-changes were calculated as relative to unstimulated cells, after GAPDH normalization. Experiments are the average of three animals, each measured in triplicate wells. All viral stimulation experiments (ICS and qPCR) were performed using AT2-inactivated preparations of SIV_mac239 or HIV-1.</p

Association of SM polymorphisms with markers of SIV disease progression.

a<p>mean and S.E. are indicated for denoted genotypes; degress of freedom are indicated by parantheses.</p>b<p>significance threshold corrected for multiple hypothesis testing (Bonferroni) is 0.01.</p

Genotype frequencies of predicted amino acid substitutions in Sooty Mangabey IRF7.

a<p>aa position in SM IRF7.</p

SIVmac239 and TLR7 agonists induce IFN-β production by SMs.

<p>PBMCs were stimulated with CL097 (A) or SIV_mac239 (B), RNA was harvested at indicated time points and quantitated by qPCR. Fold-changes were normalized by GAPDH, and are expressed as relative to unstimulated replicates. Values are averages of three animals; values for individual animals were averaged from triplicate wells. (C) PBMCs from RMs and SMs were incubated for CL097 and supernatants were harvested at the times indicated and IFN-β was assessed by ELISA.</p

Evaluation of r-mamu-IFN-α efficacy in AGMs.

<p>(A) <i>In vitro</i> comparison of the induction of ISG expression in AGM (hatched green) <i>versus</i> rhesus macaques (red) PBMCs after 18 h stimulation with 10, 100 or 1000 IU/ml of r-mamu-IFN-α (n = 2 AGMs, n = 2 macaques). <i>Mx1</i> gene expression values were measured by real-time PCR and are expressed as the Log₂ (fold change relative to values before treatment). The experiment was done in triplicate. (B) IFN-α detection in the plasma of SIV-negative (hatched green) and SIV-infected (blue) AGMs after a single subcutaneous injection of 5×10⁵ IU of r-mamu-IFN-α. IFN-α was rapidly detectable in blood (1 h), but decreased after 24 h. (C) Efficient induction of ISG (<i>CXCL10</i>, <i>IP-10</i>) in the PBMC of SIV-negative (hatched green) and SIV-infected (blue) AGMs (chronic phase) after a single subcutaneous injection of 5×10⁵ IU of r-mamu-IFN-α. Gene expression was measured by real-time PCR and expressed as the Log₂ (fold change relative to values before treatment). (D) Viral load in a chronically SIV-infected AGM treated during 16 days with r-mamu-IFN-α as described in results. (E) Impact of the r-mamu-IFN-α treatment during acute infection on viral load. SIVagm-infected untreated AGMs are shown in black and IFN-α-treated in blue. Plasma viral load are reported as log₁₀ (viral RNA copies)/ml of plasma. (F) No major effect of the two weeks IFN-α treatment on T cell proliferation in an uninfected AGM and in a chronically infected AGM (same AGM as in D) as measured by flow cytometry. (G and H) No effect of the treatment during the acute phase on T cell proliferation. T cell proliferations are shown as percentage of Ki-67⁺ cells among blood (G) CD4⁺ and (H) CD8⁺ T cells. SIVagm-infected untreated AGMs are shown in black and IFN-α-treated in blue. The grey area indicates the period of treatment. The medians of treated animals were inside the interquartile range of the control untreated animals and were thus considered not different.</p

High dose of IFN-α in the acute phase of infection does not induce persistent ISG expression in AGMs.

<p>The 6 panels show the expression levels in PBMCs and LN cells of 3 ISGs: (A,B) <i>CXCL9</i>, (C,D) <i>CXCL10</i> (<i>IP-10</i>) and (E,F) <i>CXCL11</i>. Gene expression was measured by real-time PCR and expressed as the Log₂ (fold change relative to values before treatment). IFN-α treated AGMs are in blue and untreated in black. The grey area indicates the period of treatment. The medians of treated animals were inside the interquartile range of the untreated animals and were thus considered not different.</p

Expression of homing and maturation marker on mDCs in blood and lymph nodes following SIVagm infection.

<p>MDCs were defined as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004241#ppat.1004241.s001" target="_blank">Figure S1</a>. Expression levels of the chemokine receptors (A, B) CXCR3 and (C, D) CCR7 and the co-stimulatory (F, G) CD86 and (I, J) CD80 molecules on mDCs by flow cytometry in blood (left panels) and LNs (center panels) are shown as mean fluorescence intensity (MFI) after isotype-control levels were subtracted (n = 6 AGMs). A phenotypic analysis of mDC subsets was performed on another group of 8 AGMs (right panels). CD16 was added to differentiate the subsets. The expression levels of (E) HLA-DR, (H) CD86 and (K) CD80 on CD16⁺ (in purple) and CD16⁻ (in pink) mDCs are shown as MFI. Data are presented as medians and interquartile ranges. Day zero represents the median of all the time points before infection. P-values were obtained from a linear mixed effect model to characterize each marker's progression (with a logarithm transformation for panel A and B).</p