Prospectively defined murine mesenchymal stem cells inhibit Klebsiella pneumoniae-induced acute lung injury and improve pneumonia survival

Mice, Klebsiella pneumoniae infection and MSC treatment

Specific-pathogen-free C57BL/6 (C57BL/6NCrl) and B6.Cg-Tg(TcraTcrb)425Cbn/J mice (20–25 g
each) were purchased from Charles River, Germany and maintained under specific-pathogen-free
conditions. The mice were infected intratracheally with 3.5?×?10
⁵
 CFU of Klebsiella pneumoniae (K. pneumoniae) serotype 2 (American Type Culture Collection (ATCC) 43816) in 50 ?l sterile 0.9 % NaCl as previously described 23]. Four hours p.i. the anesthetized mice received 1?×?10
⁶
washed P?S MSC in 50 ?l 0.9 % NaCl intratracheally and were analyzed at the indicated
time points (KpN/MSC). The control mice were treated identically but received 50 ?l
0.9 % NaCl (KpN/NaCl). Some mice received 1?×?10
⁶
washed mouse lung fibroblasts (MLg;ATCC CCL-206) in 50 ?l 0.9 % NaCl and were indicated
as such (KpN/MLg). Experiments were approved by the regional animal authority board
(#75/2011).

BM preparation, P?S MSC sorting and in vitro expansion

Femura and tibiae were prepared as previously described with minor modifications 7]. The bone fragments were collected and digested for 1 h at 37 °C in alpha-MEM with
L-Glutamine (PAN Biotech, Germany), 10 % FBS (PAA, Germany), 1 % penicillin/streptomycin
(PAN Biotech) containing 3.92 U/ml collagenase (Wako Chemicals, Japan), 10 mM Hepes
(Gibco, Germany) and 3 mM CaCl2. The cell suspension was filtered through a 70 ?m
cell strainer (BD Falcon, Germany) and collected by centrifugation at 400 g for 5 min
at 4 °C. Red blood cells were lysed using 155 mM NH
₄
Cl/10 mM KHCO
₃
buffer (pH 7.4) and washed with HBSS (PAN Biotech, Germany). After digestion, leucocytes
were depleted with CD45 magnetic beads (Miltenyi Biotech, Germany) and stained with
fluorochrome-labelled monoclonal antibodies and sorted by a BD ARIAIII cell sorter
(Becton Dickinson, San Jose, CA, USA). P?S MSC were defined as positive for CD140a
and Sca-1 and negative for CD45 and TER119 and were expanded in PureCoat Amine plates/flasks
(BD, Germany) in alpha-MEM medium supplemented with L-Glutamine, 5 % FBS (mesenchymal
stem cell-qualified, Life technologies, Germany), and 5 % human platelet lysate. Medium
was changed every 3–7 days depending on cell growth. Human platelet lysate was prepared
as previously described 24].

Lung preparation

Lung single cell suspensions (lung homogenates) were prepared after enzymatic digestion
as previously described in detail 23]. In brief, the mice were euthanized and the lungs were perfused via the right ventricle
with HBSS (PAA, Germany) to remove the intravascular pool of cells. The tissues were
minced and digestion was performed in 0.09 U/ml type A collagenase (Roche, Germany)
and 9.09 U/ml DNase (Roche, Germany) in IMDM (PAA, Germany) with 10 % FCS (PAA, Germany)
at 37 °C for 1 h. The single cell suspensions were prepared by tissue resuspension
with 20 G 1 ½ cannulas (0.9?×?40 mm; BD, Germany) and by mashing through a 70 ?M cell
strainer (BD, Germany). Red blood cells were lysed by ammonium chloride lysis. The
cells were washed with HBSS for flow cytometry staining, or the leukocytes were magnetic-bead
sorted after washing with PBS/2 % BSA/2 mM EDTA (PAA, Germany). Bronchoalveolar lavages
(BAL) were performed as previously described 25]. The bacterial load in lung homogenates and BAL were defined by preparing a two-fold
dilution series in sterile HBSS after centrifugation (2500 g, 15 min, 4 ° C) according
to the method developed by Schott 26].

Flow cytometry

Cellular phenotyping and sorting were performed on a BD ARIAIII cell sorter (Becton
Dickinson, San Jose, CA, USA). The following fluorochrome-labelled mAbs conjugated
to FITC, PE, PeCy7, PerCPCy5.5, APC, APC-Cy7, Brilliant Violet 510, Brilliant Violet
605, Pacific Blue and Alexa700 or appropriate isotype controls were used for cell
surface staining: CD11b, CD11c, CD45 (clone 30-F1), CD86, CD103, CD140a (clone APA5),
CD274 (PD-L1), MHC-class II (I-A
^b
), GR-1, F4/80, NK1.1, SCA-1 (clone D7), Siglec-F (BD Biosciences, Germany), TER119
and 120G8 (Dendritics, France). The surface staining time was 30 min on ice and cells
were washed with staining buffer (1× HBSS, PAA, Germany) at 400 g for 5 min at room
temperature (RT) before analysis. The number of acquired events was???500,000.

The viability of sorted cells was 90 % as indicated by Sytox blue (Life Technologies,
Germany) staining. All mAbs were ordered from Biolegend, Germany unless indicated
otherwise. Absolute cell counts were determined with AccuCount Fluorescent Particles
7.7 ?m (Spherotech, Lake Forest, USA).

CD4 T cell proliferation assay

CD4+ T cells were isolated from the spleens of OT-II mice (B6.Cg-Tg(TcraTcrb)425Cbn/J)
using the CD4+ T Cell Isolation Kit (Miltenyi Biotec, Germany). CD4+ T cells were
labeled with 0,5 ?M CFSE (eBioscience, Germany) for 15 min at 37 °C with frequent
agitation, then washed before being used in proliferation assays. DC were generated
from C57BL/6 BM hematopoietic stem cells by culturing for 7 days in 10 ng/ml murine
GM-CSF and 10 ng/ml murine IL-4 as previously described 27]. CD4+ T-cells (1?×?10
⁵
) were cultured with DC (1?×?10
⁴
) and MSC (1?×?10
⁴
) in RPMI 1640 containing 10 % FCS (PAA, Germany) for 5 days. Ovalbumin (OVA, 100 ?g/ml)
protein (Hyglos, Germany) was added to the culture medium. OVA-specific proliferation
was evaluated as a CFSE dilution by flow cytometry.

Intracellular cytokine and Foxp3 staining

For cytokine staining cells were stimulated for 6 h at 37 °C with 50 ng/ml PMA (Sigma-Aldrich,
Germany), 1 ?g/ml Ionnomycin (Sigma-Aldrich, Germany) and 3 ?g/ml brefeldin A (eBioscience,
Germany) in RPMI with 10 % FBS (PAA, Germany). For intracellular cytokine or Foxp3
staining samples were first stained for surface antigens, washed with PBS (PAN Biotech,
Germany), and centrifuged for 5 min at RT and 400 g. The cell pellets were vortexed
for dissociation and incubated with fixation/permeabilization buffer (BD, Germany)
20 min at RT. After washing twice with 2 ml of permeabilization/washing buffer (BD,
Germany) the cells were resuspended in 100 ?l permeabilization buffer. Intracellular
mAbs (IFN-?, IL-4, IL-10, IL-17 from Biolegend, Germany; Foxp3, eBioscience, Germany)
or isotype controls were added at the recommended concentrations and incubated 30 min
at RT. Cells were washed two times with permeabilization/washing buffer and immediately
analyzed by flow cytometry. The number of acquired events was???500,000.

Respiratory leukocyte subset discrimination

The gating strategy has been described recently with minor modifications 23]. Briefly, out of the CD45
⁺
cells, neutrophils were identified by GR1
^bright
CD11b
^bright
expression. Subsequently, out of the neutrophil negative fraction, macrophages were
identified as SiglecF
⁺⁺
/CD11c positive cells. DC were identified according to CD11c
⁺
Siglec-F
^neg
NK1.1
^neg
expression to exclude autofluorescent macrophages and NK cells. Then they were further
dissected into plasmacytoid DC (120 g8
⁺
CD11b
^neg
) and after MHC-class II
⁺
gating into CD103 DC (CD103
⁺
CD11b
^neg
) and CD11b DC (CD11b
⁺
CD103
^neg
). CD3
⁺
T cells and CD19
⁺
B cells were identified within the CD45
⁺
SSC
^low
fraction. Out of the CD3
⁺
T cell fraction, CD4
⁺
and CD8
⁺
T cells were identified on the basis of CD4 and CD8 expression, respectively. NK cells
were identified as CD3
^neg
NK1.1
⁺
cells and ?? T cells were identified as CD3
⁺
?? TCR
⁺
cells (Additional file 1: Figure S1). T regulatory cells were identified as CD3
⁺
CD4
⁺
CD25
⁺
Foxp3
⁺
cells.

BAL protein and cytokine quantification

Protein quantification was performed with Pierce BCA Protein Assay Kit (Thermo Scientific,
Germany). Mouse TNF-?, IL-10 and IL-12p70 were quantified by cytometric bead arrays
according to the manufacturer’s instructions (Flowcytomix, eBioscience, Germany.

Statistical analyses

Statistical analyses were performed using the GraphPad Prism software version 5.02
(Graphpad Software, Inc., USA). The significance of any differences between groups
were analyzed by the one-way ANOVA and Tukey post-test for multiple comparisons. Survival
curve comparison and analysis was performed using the logrank test. A p-value of??0.05 was considered statistically significant.