European Journal of Immunology

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Volker Brinkmann and Stefan H. E. Kaufmann. Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte ...
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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201344219 Anca Dorhoi, Vladimir Yeremeev, Geraldine Nouailles, January Weiner 3rd, Sabine Jorg, Ellen Heinemann, Dagmar Oberbeck-Muller, Julia K. Knaul, ¨ ¨ Alexis Vogelzang, Stephen T. Reece, Karin Hahnke, Hans-Joachim Mollenkopf, Volker Brinkmann and Stefan H. E. Kaufmann Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics

C 2014 The Authors. European Journal of Immunology published by WILEY-VCH Verlag

GmbH & Co. KGaA, Weinheim

www.eji-journal.eu

Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics Anca Dorhoi1,*, Vladimir Yeremeev1,2,*,Geraldine Nouailles1, January Weiner 3rd 1, Sabine Jörg1, Ellen Heinemann1, Dagmar Oberbeck-Müller1, Julia Knaul1, Alexis Vogelzang1, Stephen T. Reece1, Karin Hanke1, Hans-Joachim Mollenkopf1,3, Volker Brinkmann4, Stefan H.E. Kaufmann1

Supporting Information: Figures 1–7

CFUs (log10)

A

**

8 6

C57BL/6

129S2

B

129S2 C57BL/6

4 2 0

7

C

25

140

D

WT (129S2) Ifnar1-/-

120

lung/body weight %

Body weight change (%)

100 80 60 0

2.5

1.5 1.0 0.5 WT (129S2)

Days p.i.

WT (129S2)

Ifnar1-/-

**

2.0

0.0

7 14 22 29 35 49 56 64 71 79

Ifnar1-/-

F 4000

LDH IU/ml

E

14 Days p.i.

3000 2000 1000 0 WT(129S2)

Ifnar1 -/-

Supporting Information: Figure 1 Characteristics of TB disease in susceptible and resistant mice and in Ifnar1–/– animals. (A) Bacterial burdens in lungs of Mycobacterium tuberculosis (Mtb)-infected 129S2 and C57BL/6 mice (~200 CFUs; npooled=8-10; median±IQR). Data from two independent experiments, Mann-Whitney test for statistical analysis. (B) Histopathology of lungs, Giemsa staining of paraffin-embedded tissue collected at 25 days p.i. (scale bar, 1000 μm; n=5). Data from one representative of three independent experiments. (C) Relative body weights of WT (129S2) and Ifnar1–/– mice infected with low-dose of Mtb (~200 CFUs) (npooled=8-10; mean±SEM). Data from two experiments. (D) Relative lung weights of WT (129S2) and Ifnar1–/– mice at day 21 p.i. (npooled=8-9; mean±SEM). Data from two independent experiments. (E) Lung pathology, Giemsa staining of paraffin-embedded tissue collected at 25 days p.i. (scale bar, 1000 μm; n=5). Data from one representative of two independent experiments. (F) Serum concentrations of lactate dehydrogenase at day 21 p.i. in WT mice (129S2) and Ifnar1–/– mice (background 129S2) (npooled =11-13; mean±SEM). Data from two independent experiments. ** P < 0.01

A

B

****

100

Survival (%)

CFUs (log10)

8 7 6 5 4 WT (C57BL6)

Ifnar1-/-

80 60 WT (C57BL/6) Ifnar1-/-

40 20 0 0

20

40

60

80

Days p.i.

Supporting Information: Figure 2 TB disease in C57BL/6 and corresponding Ifnar1–/– animals. (A) Bacterial burdens (day 42) in lungs of infected WT (C57BL/6) and Ifnar1–/– mice (~500 CFUs; npooled =15– 21; median±IQR). Data from two independent experiments, Mann–Whitney test for statistical analysis. (B) Survival of WT (C57BL/6), and Ifnar1–/– mice (npooled =12–20) after aerosol infection with virulent Mtb H37Rv (~200 CFUs), Kaplan–Meier curves. Data from two independent experiments. **** P < 0.0001

CD4+ T cells

2.5

CD8+ T cells

# IFNg+ cells [x104]

% IFNg+among cells

A

2.0 1.5 1.0 0.5 0.0 14

21

14

21

CD4+ T cells

8

CD8+ T cells WT (129S2)

6

Ifnar1-/4 2 0 14

21

14

21

Days p.i.

Days p.i.

B 15

WT (129S2)

# FoxP3+CD4+ lymphocytes [x105]

% FoxP3+among CD4+ lymphocytes

Treg 20

Ifnar1-/-

10 5 0 14

Days p.i.

21

5 4 3 2 1 0 14

21

Days p.i.

Supporting Information: Figure 3 Type I IFN signaling does not modify T cell responses. WT (129S2) and Ifnar1–/– mice were infected with Mtb and leukocyte populations isolated from lungs and analyzed by flow cytometry at indicated time points. (A) Flow cytometric gating strategy used for analysis of T cells. Frequencies of IFN-γ+ cells among lung CD4+ and CD8+ T cells and numbers of IFN-γ+TNF-α+ lung CD4+ and CD8+ T cells after short-term in vitro restimulation with Mtb-derived PPD (npooled=8–10; mean±SEM). (B) Frequencies of FoxP3+ cells among lung CD4+ T cells and numbers of FoxP3+CD4+ lung lymphocytes (npooled=8–10; mean±SEM). (A–B) Mean values from two independent experiments.

R1: AM, CD11chiCD11b-

R2 Ly6G

CD11c

R2: PMN, Ly6GhiCD11bhi R3: Ly6G-/dimCD11c-/dim R4: LM, R3+ F4/80+CD11b+ R5: iM, R4+ Ly6Chi CD11b

CD11b

leukocytes gated

R3 gated

R3

R5

CD11b

CD45-

CD45 +

Lung

numbers [x105]

% among leukocytes

-/-

Ifnar1

10 5 0

E Nucleosome enrichment OD [A405]

0.3 0.2 0.1 0.0

0.5 0.4

WT (129S2) Ifnar1-/-

0.3 0.2 0.1 0.0 0

6 4 2

10

5

0

0

CD45-

0.4

WT (C57BL/6)

15

Ifnar1-/-

7

Days p.i.

11

CD45 +

Lung

KEGG Pathways

20

8

15

WT

F4/80

GO Terms

D

alveolar macrophages

alveolar macrophages 10

Ly6C

F4/80

CD11c

R4

Ly6G

C

R4 gated

B

numbers [x105]

leukocytes gated

leukocytes gated

R1

% among leukocytes

A

Supporting Information: Figure 4 Analysis of lung cell populations in WT and Ifnar1–/– mice during TB. (A) Flow cytometric gating strategy used for analysis of lung-innate cells: AMs (CD11chiCD11b–), PMNs (Ly6GhiCD11bhi), LMs (CD11c–Ly6G– F4/80+CD11b+), iMs (CD11c–Ly6G– F4/80+CD11b+ Ly6Chi). (B) Numbers and frequencies of AMs in naive WT (129S2) and Ifnar1–/– mice (n=10; mean±SEM). Data representative of two experiments. (C) GO enrichment and KEGG pathway analysis. Left: Plots show log fold changes in gene expression between naive mice and day 14 p.i. for the WT (129S2) strain (horizontal axis) and the IFNAR1 KO (vertical axis) strain; each point corresponds to one gene from the given GO category. Three-plot columns correspond to the samples tested: CD45– cells, CD45+ cells and RNA isolated from the whole lung. Genes depicted in color show significant interactions between infection and mouse strain. Red denotes genes for which log fold change is higher in the KO than in WT, and blue marks genes for which log fold change is higher in the WT than in KO. P-values correspond to significance of GO enrichment. Right: SPIA analysis of KEGG pathways. P-values correspond to the pGFWER composite p-value from the SPIA analysis. (D) PMN frequencies and numbers in BAL obtained at 14 days p.i. from C57BL/6 and corresponding Ifnar1–/– mice infected with virulent Mtb H37Rv (500 CFUs). Results are from one experiment (n=5; mean±SEM). (E) Nucleosome abundance in BALF obtained from naïve animals or at 7 and 10 days p.i. Data from two independent experiments (npooled=11; mean±SEM).

A

B

Supporting Information: Figure 5 Analysis of cell death and bacterial content within myeloid BAL cells during TB. Flow cytometric gating strategy used for (A) analysis of cell death events within BAL cells and (B) identification of Mtb-GFP cells within airways. Cells were gated using surface makers depicted in Fig. S4A to identify AMs and PMNs and subsequently dead cells were identified as annexinv+7AAD+ (A) or the GFP signal was identified using samples from naïve mice or mice infected with wild-type Mtb, lacking the GFP reporter (B).

BMDCs WT(129S2) WT + IFN-

5

B

3

-/-

Ifnar1 Ifnar1-/- + IFN-

4 3 2 1

CFUs (log10)

cpm (x 104)

A

2

1

0

0 0

50

100

WT (129S2)

150

Ifnar1-/-

hours p.i. BMDMs

WT(129S2)

8

WT + IFN-

cpm (x 104)

Ifnar1-/6

Ifnar1-/- + IFN-

4 2 0 0

50

100

150

hours p.i.

cpm (x 104)

LMs

WT(129S2)

12

WT + IFN-

10

Ifnar1-/Ifnar1-/- + IFN-

8 6 4 2 0 0

50

100

150

hours p.i.

Supporting Information: Figure 6 Mtb phagocytosis and replication in BM-derived phagocytes, lung macrophages and alveolar macrophages . (A) BMDMs, BMDCs and LMs were infected with Mtb at MOI 5 and bacterial replication was determined by [3H]uracil uptake at different time points p.i. Data from one representative of three independent experiments with three replicates each. (B) AMs were infected with Mtb at MOI 10 and bacterial uptake was estimated by CFUs at 4 hours p.i. Data from one representative of two independent experiments with three to four replicates each.

Ifnar1-/-

100

Spleen

65.5%

% GFP+ cells

WT (129S2)

65.2%

80

WT (129S2) Ifnar1-/-

60 40 20 0

Ly6G

Bone marrow

Bone marrow

27.0%

31.5%

BCG (GFP)

MFI (GFP-BCG)

5000 4000

Spleen

WT (129S2) Ifnar1-/-

3000 2000 1000 0 Bone marrow

Spleen

Supporting Information: Figure 7 Phagocytic properties of WT (129S2) and Ifnar1–/– cells for mycobacteria. Representative FACS plots showing phagocytosis of GFP-BCG by Ly6G+ PMNs purified from BM and spleen from WT (129S2) and Ifnar1–/– mice, two independent experiments with two to three replicates each.