A Petri Net Model of Granulomatous Inflammation: Implications for IL-10 Mediated Control of <i>Leishmania donovani</i> Infection

<div><p>Experimental visceral leishmaniasis, caused by infection of mice with the protozoan parasite <i>Leishmania donovani</i>, is characterized by focal accumulation of inflammatory cells in the liver, forming discrete “granulomas” within which the parasite is eventually eliminated. To shed new light on fundamental aspects of granuloma formation and function, we have developed an <i>in silico</i> Petri net model that simulates hepatic granuloma development throughout the course of infection. The model was extensively validated by comparison with data derived from experimental studies in mice, and the model robustness was assessed by a sensitivity analysis. The model recapitulated the progression of disease as seen during experimental infection and also faithfully predicted many of the changes in cellular composition seen within granulomas over time. By conducting <i>in silico</i> experiments, we have identified a previously unappreciated level of inter-granuloma diversity in terms of the development of anti-leishmanial activity. Furthermore, by simulating the impact of IL-10 gene deficiency in a variety of lymphocyte and myeloid cell populations, our data suggest a dominant local regulatory role for IL-10 produced by infected Kupffer cells at the core of the granuloma.</p></div

IRF7 Regulates TLR2-Mediated Activation of Splenic CD11c^hi Dendritic Cells

<div><p>Members of the Interferon Regulatory Factor (IRF) family of transcription factors play an essential role in the development and function of the immune system. Here we investigated the role of IRF7 in the functional activation of conventional CD11c^hi splenic dendritic cells (cDCs) <em>in vitro</em> and <em>in vivo</em>. Using mice deficient in IRF7, we found that this transcription factor was dispensable for the <em>in vivo</em> development of cDC subsets in the spleen. However, IRF7-deficient cDCs showed enhanced activation in response to microbial stimuli, characterised by exaggerated expression of CD80, CD86 and MHCII upon TLR2 ligation <em>in vitro</em>. The hyper-responsiveness of <em>Irf7</em>^−/− cDC to TLR ligation could not be reversed with exogenous IFNα, nor by co-culture with wild-type cDCs, suggesting an intrinsic defect due to IRF7-deficiency. <em>Irf7</em>^−/− cDCs also had impaired capacity to produce IL-12p70 when stimulated <em>ex vivo</em>, instead producing elevated levels of IL-10 that impaired their capacity to drive Th1 responses. Finally, analysis of bone marrow microchimeric mice revealed that cDCs deficient in IRF7 were also hyper-responsive to TLR2-mediated activation <em>in vivo</em>. Our data suggest a previously unknown function for IRF7 as a component of the regulatory network associated with cDC activation and adds to the wide variety of situations in which these transcription factors play a role.</p> </div

Exaggerated CD86 expression after TLR2 stimulation occurs in the presence of IRF7-sufficient cDCs or exogenous IFNα.

<p>A. Representative purity of splenic CD11c^hi cDCs sorted from C57BL/6, B6.<i>Irf7</i>^−/− and congenic B6J.CD45.1 mice. B. Representative staining of cDCs from B6.<i>Irf7</i>^−/− and B6J.CD45.1 mice after co-culture at <b>∼</b>50∶50 ratio in the presence of 10 µg/ml PAM₃CSK₄. C. The fold increase in expression of CD86 after TLR2 stimulation for cDCs from either strain was determined by flow cytometry at the indicated times post-stimulation. D. C57BL/6 or B6.<i>Irf7</i>^−/− cDCs were cultured in the presence of 10 µg/ml PAM₃CSK₄ and 1000 U/ml IFNα. At the indicated times post- stimulation, cells were removed and assessed by flow cytometry expression of CD86. Data are presented as mean fold increase ± SEM in surface expression of CD86 relative to unstimulated cDCs from the same strain. Data are from two experiments. * = p<0.05 ** =  p<0.01, *** = p<0.001.</p

IRF7-deficient cDCs show hyper-responsiveness to PAM₃CSK₄<i>in vivo</i> even in an IRF7-sufficient environment.

<p>B6.<i>Irf7</i>^−/− or C57BL/6 microchimeric mice were generated by bone marrow transfer into Busulfan-treated congenic B6J.CD45.1 hosts. <b>A</b>. 7 to 14 days post- engraftment, microchimeric mice bearing B6.<i>Irf7</i>^−/− or C57BL/6 chimeric cell populations received 5 µg/mouse PAM₃CSK₄ intravenously. Splenic CD11c^hiMHCII^hi cDC compartments in microchimeric animals were comprised of endogenous (CD45.1⁺) and chimeric (CD45.1^-) cell populations (<b>B</b>). After 24 hours, activation of splenic cDCs after PAM₃CSK₄ administration <i>in vivo</i> was assessed by quantifying changes in surface expression of CD80 (<b>C</b>) and CD86 (<b>D</b>) by flow cytometry. Flow plots in <b>B</b> are representative. Data in <b>C</b> and <b>D</b> show the mean fold change in indicated surface protein ± SEM on endogenous (open bars) and chimeric (black bars) cDCs after PAM₃CSK₄ injection, compared to the same populations of cDCs in mice receiving PBS. n = 4 per group, representative of two experiments. ** =  p<0.01.</p

Schematics of the model dynamics.

<p>(<b>A</b>) High-level depiction of the interactions among the entities modeled. (<b>B</b>) Differentiation of helper T cells. Labels on arrows indicate the conditions for differentiation. Arrows pointing to/originating from a cytokine name indicate that the cytokine is produced/consumed by the cell. (<b>C</b>) Differentiation of cytotoxic T cells. Arrow conventions as in panel B. (<b>D</b>) Dynamics of activation types in macrophages. <i>Leishmania</i> interactions are restricted to Kupffer cells only. Note how different cytokines promote different types of activation and how different types of activation result in the production of different cytokines. (<b>E</b>) Differentiation of NK cells. Arrow conventions as in panel B. (<b>F</b>) Transitions from/to inactive to/from active states for the modeled leukocytes. This representation stresses the complexity of the model and the degree of interaction among the different cell populations; see Section 1 of Supplementary Information for a more detailed description.</p

IRF7-deficient splenic cDCs are hyper-responsive to a TLR2 agonist <i>in vitro</i>. A

<p>. CD11c^hi cDCs were sorted to <b>∼</b>99% purity from the spleens of C57BL/6 and B6.<i>Irf7</i>^−/− mice. Cells were cultured in triplicate at 1×10⁶ cells/ml in the presence of 10 µg/ml PAM₃CSK₄. At the indicated times post-stimulation, cells were removed and assessed by flow cytometry for expression of CD80, CD86 and MHCII. Representative flow plots showing progressive cDC activation in terms of CD86 and MHCII expression are shown in <b>B</b>, fold increases in surface expression of CD80, CD86 and MHCII on cDCs at the indicated time point over unstimulated cDCs are shown in <b>C</b>, <b>D</b> and <b>E</b>, respectively. <b>C</b>–<b>E</b> show mean fold increase ± SEM in surface expression of indicated proteins on cDCs from C57BL/6 (open bars) or B6.<i>Irf7</i>^−/−(closed bars) mice, compared to unstimulated cDCs from the same strain. Data are pooled from three individual experiments. * = p<0.05 ** =  p<0.01, *** = p<0.001.</p

<i>In silico</i> cell-specific knock out of IL-10 implicates Kupffer cell IL-10 production as a major determinant of leishmanicidal activity within granulomas.

<p>(<b>A</b>) <i>In silico</i> knockout of IL-10 in mononuclear phagocytes (Mono IL-10⁻), T cells (T IL10⁻), and NK cells (NK IL10⁻) compared with baseline <i>in silico</i> model and in vivo (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003334#pcbi.1003334-Murray5" target="_blank">[58]</a>; WT). (<b>B</b>) <i>In silico</i> knockout of IL-10 from Kupffer cells (KC IL10⁻) and non-resident macrophages/monocytes/DC (Mac IL10⁻), compared with baseline <i>in silico</i> model and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003334#pcbi.1003334-Murray5" target="_blank">[58]</a>. In all the panels, means and standard deviation are reported. Standard deviation is indicated by error bars or shaded areas.</p

Simulations reflecting gene KO qualitatively reproduce expected changes in disease outcome.

<p>(<b>A</b> and <b>B</b>) Parasite burden after <i>in silico</i> knock out of T cells (A) or IFNγ (B), compared to the results from the baseline model (baseline) and in vivo (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003334#pcbi.1003334-Murray5" target="_blank">[58]</a>; WT). (<b>C</b>) <i>In silico</i> knock out of IL-10 compared with <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003334#pcbi.1003334-Murray5" target="_blank">[58]</a> and data adapted from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003334#pcbi.1003334-Stanley1" target="_blank">[61]</a>). In all the panels, means and standard deviation are reported. Standard deviation is indicated by error bars or shaded areas.</p

Baseline model allows the exploration of biological quantities difficult to access experimentally.

<p>In all the panels, means and standard deviation (indicated by shaded area around the mean) are reported. All numbers are relative to cells in the liver associated with a granuloma microenvironment. (<b>A</b>) Number of granuloma-associated non-resident macrophages. (<b>B</b>) Number of differentiated Th1 cells. (<b>C</b>) Number of activated NK cells. (<b>D</b>) Level of activation and deactivation of non-resident macrophages. (<b>E</b>) Level of activation and deactivation of Kupffer cells. (<b>F</b>) Number of activated NKT cells. (<b>G</b>) Concentration of IFNγ and IL-10. (<b>H</b>) Concentration of IL-2and IL-12. (<b>I</b>) Concentration of IL-4 and IL-10.</p

Effects of Ly6G depletion on cervico-vaginal IgG.

<p>Naïve virgin C57BL/6 mice were treated with the Ly6G-depleting antibody 1A8 or the isotype control 2A3 at PE (<b>A</b>), E (<b>B</b>), ME (<b>C</b>) or DE (<b>D</b>). Cervico-vaginal washings were taken 24 and 48h after mAb administration according to the described protocol. Data are presented as ratios of genital IgG levels post-injection, measured by ELISA, relative to genital IgG levels pre-injection (baseline). Data are derived from one experiment (n≥4 mice) and were analyzed using unpaired t test. The mean±SEM is shown (n.s., non-significant).</p