Pathogens Improve Their Survival by Overcoming INS Immune Defenses When Targeting TLRs or “Danger” Immune Defenses When Targeting Apoptosis

<div><p>Microbes undertake two major strategies to overcome the host immune system: interfering directly with APC's pattern recognition receptors (INS model side), or triggering apoptosis of phagocytes that in turn inhibits proximal APCs (danger model side). Both strategies ultimately lead to tolerance by inducing presentation of antigen in absence of crucial co-stimulatory molecules.</p><p>Viruses have been shown to have a myriad of strategies to disrupt the TLR cascade, mainly focusing on the IFN response, while bacteria act far downstream on mitogen-activated kinase kinases and NF-κB.</p><p>Pathogen-induced cell death induces the release of cellular blebs expressing phosphatidylserine (PS) that are rapidly internalized by neighboring cells or phagocytes. Infected, apoptotic cells may send these NSs to limit the effectiveness of antigen presentation by neighboring uninfected APCs to T helper cells. In this situation, without co-stimulatory molecules and secreted IL-12 but in presence of IL-10 and transforming growth factor β, ultimately tolerance to microbial antigens will be induced.</p><p>AP1, activator protein 1; GC-BP, GC binding protein; IFN, interferon; IL, interleukin; IRAK, interleukin-1-associated kinase; IRF, IFN-regulatory factor; MAPK, mitogen-activated protein kinase; MHC, major histocompatibility complex; MKK, MAPK kinase; MyD88, myeloid differentiation primary-response factor 88; NF-κB, nuclear factor κB; PSR, phosphatidylserine receptor; TAK, TGF-β-activated kinase; TBK, TRAF-family-member-associated NF-κB activator-binding kinase; TCR, T cell receptor; TGF, transforming growth factor; TLR, Toll-like receptor; TRAF, TNF-receptor-associated factor; TRAM, TRIF-related adaptator molecule; TRIF, TIR-domain-containing adaptor inducing IFN-β</p></div

ET and LT modulate MoDCs cytokines secretions.

<p>MoDCs were infected with different strains of <i>B. anthracis</i>: RP42 (LT−/ET−), Sterne (LT+/ET+), RP9 (LT+/ET−), or RP10 (LT−/ET+) (A); or pre-treated with purified toxins, and then infected with the non-toxinogenic RP42 strain (B). Data show mean Il-12p70, IL-10, and TNF-α concentrations in culture supernatants (+/−SD) representative of three independent experiments. (C) MoDCs were pre-treated with a different range of purified ET concentrations (1, 10 and 40 ng/ml), and then infected with the non-toxinogenic RP42 strain. Data show mean Il-12p70, IL-10, and TNF-α concentrations in culture supernatants (+/−SD) representative of three independent experiments. Student <i>t-test</i>; <i>*, p<0.05; **, p<0.001</i> as compared with RP42 infected cells. NI: not infected.</p

Phenotype of CX₃CR1-GFP cell subsets in the lung.

<p>A: Total lung cells of CX₃CR1^+/gfp mice were gated on CX₃CR1 and analyzed for NK1.1, CD3e, CD11c, and CD11b expressions. B: Autofluorescence, CD80 and MHCII expressions on gate G1, G2 and G3 of panel A. Black histogram, isotype control; grey histogram, positive staining. C: Total lung cells were pre-gated on CD11c+ low autofluorecent cells and analyzed for the expression of CD11b and CD103. The expression of CX₃CR1 is shown on the left panel for gate G4 (CD11b⁻CD103⁺ DCs, grey histogram) and for gate G5 (CD11b⁺CD103⁻ DCs, black line). D: Total cells were pre-gated on CD45 cells and analyzed for the expression of F4/80 and CD11c. Expression of CX₃CR1 and CD11b is shown on the left panels for gate G6 (CD11c^lowF4/80^high), G7 (CD11c^highF4/80^high) and G8 (CD11c^highF4/80^low).Data from flow cytometry, performed on one CX₃CR1^+/gfp mouse lung harvested 30 minutes after intratracheal PBS injection. Data are representative of two distinct experiments.</p

Velocity of CX₃CR1-GFP positive pulmonary Dendritic-shaped and Round-shaped cells.

<p>A: Dendritic-shaped cells and B: Round-shaped cells at an early stage (average values from 1h30 to 2h30 post injection, closed symbols) and a late stage (average values from 5h to 6h post injection, open symbols) after injection of PBS (rounds) or LPS (squares). Three mice in each group, one symbol by cell. * for p<0.05; ** for p<0.01; *** for p<0.0001; ns for not significant.</p

Parameters used for individual cell analysis.

<p>A: Edge detection of two CX₃CR1+ pulmonary cells and their roundness coefficient. Scale bar  = 10 µm. B: The relevant parameters used in this work are: i) the Mean Roundness Coefficient (MRC), calculated for each cell by meaning Instantaneous Roundness Coefficient (IRC) at each consecutive observable time; ii) the Maximal Distance (MD) of a cell (red arrow) is the longest distance covered from the first position; iii) the Meandering Index (MI) is the final distance from the first position <i>D_n</i> divided by the total length covered.</p

Schematic representation of the drift correction strategy.

<p>A: Maximum intensity projection of a lung slice z-stack. Pulmonary CX₃CR1-GFP cells (green) and alveolar collagen mesh detected by collection of SHG signal (gray). Two-photon excitation wavelength  = 896 nm. B: Dashed red squares show the optic field imaged by the microscope (CX₃CR1-GFP in green and SHG in grey). The realignment phase consists in calculating the tissue drift using the maximization of SHG signal cross-correlation.</p

Maximal Distance of CX₃CR1-GFP positive pulmonary Dendritic-shaped and Round-shaped cells.

<p>A: Dendritic-shaped cells and B: Round-shaped cells at an early stage (average values from 1h30 to 2h30 post injection, closed symbols) and a late stage (average values from 5h to 6h post injection, open symbols) after injection of PBS (rounds) or LPS (squares). Three mice in each group, one symbol by cell. C, D: Overlay of Round-shaped cell tracks after late PBS (C) and LPS injection (D), after aligning their first coordinates. One color by track. Values of black circle radii in µm, equal to average cell Maximal Distance, are indicated ± standard deviation. * for p<0.05; ** for p<0.01; *** for p<0.0001; ns for not significant.</p

Discrimination of two CX₃CR1-GFP cell subsets using flow cytometry or roundness.

<p>A: Expression of CX₃CR1 <i>vs</i> CD45 receptors in pulmonary cells and CD11c <i>vs</i> CD11b by CX₃CR1^high cells. Data from flow cytometry, performed on one CX₃CR1^+/gfp mouse lung harvested 30 minutes after intratracheal PBS injection. Data representative of two distinct experiments. B: Frequency distribution of Mean Roundness Coefficients (MRC) over 1h of CX₃CR1+ pulmonary cells in lung slices harvested from 6 mice, 1h30 (3 mice) and 5h (3 mice) after PBS injection. N = 406. Cells are followed for a mean time of 32 minutes, corresponding to 16 frames. Dashed line: sum of two Gaussian fitting the histogram. R² = 0.89.</p

Network of cytokine dependence of NK cell activation by <i>B. anthracis</i> spores.

<p>Effect of neutralization of IL-12, IL-18 or IL-15Rα on (<b>A</b>) IL-12p40/p70 concentration in culture supernatants of FIS-stimulated BMDMs or (<b>B</b>) IFN-γ production by splenocytes (SPL; left panel), or purified CD49b⁺ cells co-cultured with BMDMs (right panel). (<b>C</b>) Splenocytes (SPL) from wild-type (WT), IL-12R^−/− or IL-12^−/− C57BL/6 mice were stimulated with FIS, or ConA as a positive control. (<b>D</b>) CD49b⁺ cells from WT C57BL/6 mice were co-cultured with BMDMs from IL-12R^−/− or IL-12^−/− C57BL/6 mice in the presence of FIS. (<b>E</b>) CD49b⁺ cells from WT or IL-12R^−/− C57BL/6 mice were co-cultured with BMDMs from WT C57BL/6 mice in the presence of FIS with or without IL-18 neutralizing antibody. (<b>F</b>) Effect of short-term priming with IL-12 or IL-18 on spore-stimulation of splenocytes; corresponding cytokine neutralization was maintained for the remainder of the assay. (<b>G</b>) Purified CD49b⁺ cells from WT or MyD88^−/− C57BL/6 mice were co-cultured with BMDMs from WT C57BL/6 in the presence of FIS. (<b>H</b>) Purified CD49b⁺ cells from C57BL/6 WT mice were co-cultured with BMDMs from WT or MyD88^−/− C57BL/6 mice in the presence of FIS; IL-12 (left panel), or IFN-γ (right panel) production. For all experiments with purified CD49b⁺ cells, no IFN-γ was detected after direct stimulation with spores (<b>D, E, G, H</b>; data not shown). For all experiments, values are mean ± SD for at least three measurements and are representative of at least three independent experiments. Significant differences between experimental conditions are indicated with asterisks (t test; *, <i>P</i><0.05; **, <i>P</i><0.01).</p

Recruitment and role of NK cells during <i>B. anthracis</i> infection and impact of <i>in vivo</i> toxin production.

<p>(<b>A</b>) Circulating NK cells 5 h post-inoculation, viewed by biphoton imaging; dermal collagen in blue (SHG), vascular flow in red (rhodamine B) and NK cells in green (CFSE); scale bar = 20 µm; time-scale in milliseconds indicated on each image. (<b>B</b>) Adherent, then rolling NK cell 5 h post-inoculation; scale bar = 20 µm; time-scale in seconds indicated on each image. (<b>C</b>) Extravasated NK cells at 18 h post-inoculation; scale bar = 10 µm (top), 40 µm (bottom). (<b>D</b>) Subcapsular NK cell in the cervical lymph node draining the infected ear 18 h post-inoculation; NK cells in green (CFSE) and capsular collagen in blue (SHG); scale bar = 20 µm. Data representative of 3 mice. (<b>E</b>) Absolute numbers of CD49b⁺ (left panel) and F4/80⁺ cells (right panel) in the cervical lymph node draining the site of cutaneous infection with spores of the 9602P(PA−EF+LF+), 9602L(PA+EF+LF−), 9602C(PA+EF−LF+) strains 24 h post-inoculation (2.91±0.03 log₁₀ CFU per mouse). Controls were injected with PBS. Each symbol represents the value for an individual mouse; horizontal lines indicate the mean value for each group. Data are pooled from two independent experiments. T test; **<i>P</i><0,01 as compared with the 9602P-injected group. (<b>F</b>) <i>In vivo</i> effect of NK cell depletion on systemic bacterial dissemination in the spleen. Bacterial load was determined 18 h after infection into the ear with spores of the 9602P strain (3.05±0.29 log₁₀ CFU per mouse). Data are pooled from two independent experiments.T test; *, <i>P</i><0.05; **, <i>P</i><0,01 as compared with the non-treated group.</p