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Supplementary Data
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Supplementary Figure S1. Mast cell inhibition of active caspase-3 expression and
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intramacrophage LVS infection is partially dependent on STAT6 and IL-4
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production. Mast cells, macrophages, or co-cultures were infected (MOI=100) with
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LVS followed by analysis at 3 and 24 h for caspase-3 expression and bacterial growth.
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(A) Caspase-3 expression in STAT6+/+ macrophages compared to co-cultures of
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STAT6+/+ macrophages + STAT6+/+ mast cells and co-cultures of STAT6+/+ macrophages
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+ STAT6-/- mast cells. * p < 0.03, **p < 0.002 (3-way ANOVA). (B) Bacterial growth in
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STAT6+/+ macrophages compared to co-cultures of STAT6+/+ macrophages + STAT6+/+
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mast cells and co-cultures of STAT6+/+ macrophages + STAT6-/- mast cells. *p < 0.03,
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**p < 0.003 (3-way ANOVA). (C) IL-4 and TNF-α production from STAT6+/+ or
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STAT6-/- mast cells was analyzed by ELISA at 24h.
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Supplementary Figure S2. Macrophages exhibit reduced morphological alterations
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with rIL-4 treatment during F. tularensis LVS infection. Macrophages were
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untreated, or rIL-4 treated (5 or 25 ng/ml) macrophages and infected (MOI=100) with
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Lucifer yellow-labeled LVS or mock infected. Cellular morphology was analyzed at 24
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h. Representative images of uninfected (mock) and LVS infected macrophages (CD11b,
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red) are shown with nuclear stain (DAPI, blue), and Lucifer labeled LVS (yellow).
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Magnification 400X.
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25
Wild-type Macrophages
MR-/- Macrophages
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Lucifer labeled LVS, CD11b, Acidification Probe
400 X 1 h
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acidified regions, but notable levels of bacteria with rIL-4 treatment. Wild-type and
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MR-/- macrophages untreated or rIL-4 treated were infected (MOI=100) with Lucifer
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yellow-labeled LVS. Representative images of rIL-4 treated LVS (yellow) infected WT
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and MR-/- macrophages (CD11b, green) are shown with acidification regions (red);
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Magnification 400X.
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Supplementary Figure S3. Mannose receptor deficient macrophages exhibit
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Enlargement
Enlargement
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Supplementary Figure S4. IL-4 receptor deficient macrophages exhibit minimal
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acidification regions in co-culture with mast cells compared to WT macrophages in
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co-culture with mast cells. IL-4R-/-macrophage-WT mast cell co-cultures and WT
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macrophage-WT mast cell co-cultures were infected (MOI=100) with Lucifer labeled
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LVS (yellow) for 1 h. Representative images of macrophages (CD11b, green) and mast
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cells (white arrows, unstained cell surface with blue nuclei) in co-culture are shown.
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Mast cells are shown with numerous acidified (red) organelles surrounding blue nuclei;
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Magnification 400X. Enlargements of co-cultures (IL-4R-/-macrophage-WT mast cell
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and WT macrophage-WT mast cell) are shown.
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40
Nitrite (u M )
A
30
20
10
0
*
CF U/well
6
B 10
10
*
3 h
5
24 h
1 04
1 03
1 02
Mock Unt reat ed
rIL4
25 ng/ ml
Mock
LVS
rIF N
1 0 n g / ml
Mast cel l
spent supernat ant
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Supplementary Fig. S5. Nitric oxide is not enhanced with rIL-4 or mast cell
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supernatant. LVS-infected (MOI=100) macrophages were treated with filtered mast cell
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spent supernatant, rIL-4 or rIFN-γ.
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cells analyzed for nitric oxide and bacterial growth. (A) Macrophages cultured with
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filtered spent supernatant from mock or LVS infected (MOI=100) mast cells, or rIL-4 (25
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ng/ml) were compared to untreated LVS infected macrophages for nitric oxide
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production at 24 h; Positive control (rIFN-γ,10ng/ml) shown right of the black bar. (B)
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Bacterial replication in macrophages cultured with filtered spent supernatant from mock
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or LVS infected (MOI=100) mast cells, or rIL-4 (25 ng/ml) were compared to untreated
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LVS infected macrophages at 3 h or 24 h; Positive control (rIFN-γ,10ng/ml) shown right
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of the black bar; *p < 0.05 (Unpaired t Test).
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independent experiments.
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Cultures were infected with LVS (MOI=100) and
Results are representative of three
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Supplementary Figure S6. Mast cell secreted factors inhibit intramacrophage LVS
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replication and active caspase-3.
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treated with neutralizing anti-IL-4 (25 ug/ml), anti-TNF-α (25 ug/ml) or both. Cultures
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were infected (MOI=100) with LVS and cells analyzed for active caspase-3 and bacterial
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replication. Co-culture macrophages were analyzed within the CD11b gate for flow
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cytometry and cell lysates were plated for bacterial enumeration at 3 and 24h as
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previously described. Active caspase-3 expression or bacterial replication of treated co-
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cultures were compared to untreated co-cultures; *p < 0.05 (Unpaired t Test).
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Mast cell-macrophage co-cultures were untreated,
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Supplemental Methods
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Mice
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Specific pathogen-free 4-8 week old mice were utilized for all procedures. C57BL/6 and
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BALB/c mice were purchased from the National Cancer Institute. BALB/c IL-4R-/-
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mice1 were purchased from Taconic Farms, and STAT6-/- mice were from the UT Health
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Science Center at San Antonio. All experimental procedures and animal care were
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performed in accordance with the Institutional Animal Care and Use Committee
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(IACUC) guidelines.
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Nitric Oxide Assay. Nitrite concentrations in culture supernatants were analyzed using
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the Greiss reaction as previously described2. Briefly, supernatant were incubated with
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1% sulfanilamide and 1% naphthylethylenediamine dihydrochloride (in 2.5% phosphoric
93
acid) at room temperature for 10 minutes. Nitrite was quantified (uQuant) at 545nm
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using NaNO2 standards.
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Phagocytosis assay. Phagocytosis assays were performed on wild-type or STAT6
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deficient cells (bone marrow derived mast cells or macrophages) as discussed in the
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Materials and Methods sections with modifications. On the day before the assay, mast
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cells were plated, rested 4 h, and infected (100=MOI) or mock treated for 2 h followed by
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gentamicin treatment for 1 h. The media was replaced, and the cultures were incubated
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for the remaining 24 h.
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(0.22μm), and placed on ice for use the same day as spent-culture medium. For culture
At 24 h, the supernatants were collected, syringe filtered
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analyses, murine recombinant IL-4 (25 ng/ml, eBioscience), recombinant interferon-
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gamma (rIFN-γ, 10 ng/ml) or spent supernatant, was added to cultures 2 h prior to
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infection, and replaced to cultures after each wash. Analyses were performed at 3 and 24
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h for determination of bacterial burden; and supernatant was collected at 24h and stored
107
at -80°C for cytokine or nitric oxide analyses. In separate experiments, co-cultures were
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untreated, or treated with anti-IL-4, anti-TNF-α or both for two hours prior to LVS
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infection (100=MOI). Antibodies (anti-IL-4, anti-TNF-α) were replaced after each wash
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and cells were analyzed for bacterial replication and active caspase-3 at 3 and 24 h.
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Microscopy Analysis. On the day of the assay, cells were counted and seeded (2.5 X
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105) on coverslips placed in a 24-well plate, rested 4 h, and infected (100=MOI) with
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unlabeled or Lucifer labeled LVS. Cultures were treated as described in the Methods
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section for confocal microscopy. At 1, 3 or 24 h, cells were washed with 1XPBS and
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stained as previously described. Acquisition was performed using a Zeiss 510 Meta and
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analyzed with IMARIS® software.
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References
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1
Noben-Trauth, N. et al. An interleukin 4 (IL-4)-independent pathway for CD4+ T
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cell IL-4 production is revealed in IL-4 receptor-deficient mice. Proc. Natl. Acad.
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Sci. U S A 94, 10838-10843 (1997).
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2
Green, L. C. et al. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.
Anal. Biochem 126, 131-138 (1982).