Research ArticleSystems Immunology

Coordinate actions of innate immune responses oppose those of the adaptive immune system during Salmonella infection of mice

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Science Signaling  12 Jan 2016:
Vol. 9, Issue 410, pp. ra4
DOI: 10.1126/scisignal.aaa9303
  • Fig. 1 Rapid accumulation of innate cell types in the spleen precedes the adaptive immune response.

    (A to C) Splenocytes from mice infected with 5 × 108 Salmonella bacteria were prepared for flow cytometry and analyzed by SPADE. Each bubble represents a group of cells defined by a specific lineage marker expression profile and is called a cell circle. The coloring of each cell circle represents the surface marker staining intensity (A and B) or the change in cell frequency (C) from five mice sacrificed at each time point over the 29-day time course. Each plot was generated from an equal number of cell events merged in silico from five mice. The number of cells in each cell circle is represented by the size. (A) In the SPADE plot, cell circles are colored by CD8 staining intensity, demonstrating the grouping of cell circles into cell types by lineage-specific markers (represented by a magenta box). (B) Cell circles are colored by Ly-6C staining intensity, as an example of a gradient of phenotypic marker intensity across a cell type (represented with a black arrow). (C) This figure uses color to show the change in cell frequency across the infection time course. Cell circles are colored by the change in the frequency of cells occurring in each circle at an infection time point compared to those in uninfected mice. The formula used to color cell circles was as follows: log10(percentage of cells in the cell circle during infection/percentage of cells in the cell circle in uninfected mice). An increased frequency is indicated by orange, no change by olive, and a decrease by purple. pDC, plasmacytoid dendritic cell.

  • Fig. 2 Salmonella infection induces variability in the signaling responses of splenocytes to cytokines.

    (A) Splenocytes from 12 mice infected with Salmonella for 30 days and from 4 uninfected mice were left unstimulated (unstim) or were stimulated for 15 min with IFN-γ, IL-6, IL-21 (all at 40 ng/ml), or IL-10 (80 ng/ml) before being prepared for flow cytometric analysis. The heat map shows the MFIs of pSTAT1 and pSTAT3 for the listed cell types. Mem/eff, memory/effector. (B) Correlation between the IL-6–induced phosphorylation of STAT3 in B cells and the IFN-γ–induced phosphorylation of STAT1 in effector/memory CD4+ T cells.

  • Fig. 3 Transmission status and splenic neutrophil frequency share a similar correlation pattern during Salmonella infection.

    (A) The map of correlations that reversed directions between uninfected and infected mice (20 uninfected mice and 19 mice infected for 30 to 35 days). Green circles are nodes with four or more reversals. Blue lines represent negative correlations during infection that were positive in uninfected mice, and red lines represent positive correlations during infection that were negative in uninfected mice. (B) The correlation map of infection shows all correlations with fecal bacterial load (feces CFU) and splenic neutrophil frequencies (Gr1) (filled magenta circles) obtained from data from 19 mice infected for 30 to 35 days. Red lines signify positive correlations, and blue lines signify negative correlations. Outlined blue or red circles share correlations with either fecal CFU or splenic neutrophil frequency, solid blue circles share negative correlations with both, and gray circles are not correlated with either. int, intermediate; hi, high; cDC, conventional dendritic cell; Macs, macrophages.

  • Fig. 4 Discovered connectivity shares relationships with the known regulatory network.

    This subset of network relationships from the Salmonella-infected murine system illustrates the overlap between known relationships and those reported in this work. Lines with arrowheads show previously reported causal relationships as determined through experimental intervention. The solid red and blue arrows represent known positive and negative relationships, respectively, that were present in our correlation data; the dotted blue arrow is a known relationship that was not found. Thick lines without arrowheads indicate previously uncharacterized (unreported) correlations reported here.

  • Fig. 5 Clustering of the Salmonella infection correlation matrix reveals functional cassettes comprised of attributes with shared correlations.

    (A) The correlation network from 19 infected mice shown in fig. S2B is given in matrix form. The matrix was clustered and attributes that clustered together (black boxes) were identified and annotated on the basis of function. Red represents a positive correlation, white represents no correlation, and blue represents a negative correlation. The gray box highlights the intersection of two clustered cassettes. (B) The average value within a cluster is represented colorimetrically from blue (negative) to red (positive). The rectangle at the intersection of two cassettes represents the relation between the two cassettes. (C) A pictorial representation of the relationships between the cassettes. A red line represents a positive correlation, whereas a blue line represents a negative correlation.

  • Fig. 6 Uninfected mice exhibit co-regulation of adaptive, but not innate, immunity markers.

    (A) The correlation network data obtained from 20 uninfected mice in fig. S2A is shown in matrix form. The matrix was arranged in the same order as that for infected mice in Fig. 5. n.s., not significant. (B) Representation of the relationships between cassettes.

  • Fig. 7 Neutrophils regulate STAT signaling in B cells.

    (A) Uninfected and 30-day Salmonella-infected non–super-shedder mice were left untreated or were given 1 μg of G-CSF intraperitoneally daily for 3 days before being sacrificed on the fourth day. Data are means ± SD of four mice per group. The splenocytes were left untreated or were treated with IL-6 (40 ng/ml) or IL-10 (80 ng/ml) for 15 min. Fixed and permeabilized cells were stained for phenotypic markers, and the relative abundances of pSTAT1 and pSTAT3 in B cells were quantified by flow cytometric analysis. *P < 0.05 and **P < 0.001, two-sided t test. (B) Infected mice were treated with PBS as a control or were treated with 1 μg of anti–Ly-6G (clone IA8) or anti-Gr1 (clone RBC-8C5) antibody intraperitoneally daily for 3 days and then were sacrificed on the fourth day. Data are means ± SD of three mice per group. The splenocytes were left untreated or were treated with IL-6 (40 ng/ml) for 15 min. Fixed and permeabilized cells were stained for phenotypic markers, and the relative abundances of pSTAT1 and pSTAT3 in B cells were quantified by flow cytometric analysis. *P < 0.05 and **P < 0.001, two-sided t test.

Supplementary Materials

  • Supplementary Materials for:

    Coordinate actions of innate immune responses oppose those of the adaptive immune system during Salmonella infection of mice

    Andrew N. Hotson, Smita Gopinath, Monica Nicolau, Anna Khasanova, Rachel Finck, Denise Monack, Garry P. Nolan*

    *Corresponding author. E-mail: gnolan{at}stanford.edu

    This PDF file includes:

    • Fig. S1. The adaptive immune response to Salmonella increases through the first month of infection.
    • Fig. S2. The connectivity of the immune network is increased during Salmonella infection.

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    Citation: A. N. Hotson, S. Gopinath, M. Nicolau, A. Khasanova, R. Finck, D. Monack, G. P. Nolan, Coordinate actions of innate immune responses oppose those of the adaptive immune system during Salmonella infection of mice. Sci. Signal. 9, ra4 (2016).

    © 2016 American Association for the Advancement of Science

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