Ethics statement
Use of animals and experimental procedures were reviewed and approved by the Bioethics
Committee of our Institute following established protocols.
Isolation of glycolipids
M. fortuitum ATCC 6841 was used to isolate DAT and TAT, which are similar in M. fortuitum and Mtb. Non-covalently linked lipids were extracted from live bacilli using CHCI
3
/CH
3
OH (1:2, vol/vol) and CHCI
3
/CH
3
OH (2:1, vol/vol). Pooled extracts were dried and suspended in CHCl
3
/CH
3
OH/H
2
O (4:2:1, vol/vol/vol). After that, crude lipid extracts were dissolved in chloroform
and applied to a Florisil column (Biotecna Corp., Miami, FL, USA). The elutions were
performed with chloroform and methanol and fractionation of lipids was monitored by
thin-layer chromatography (TLC) on silica gel-60 F
254
coated plates (E. Merck, Darmstadt, Germany) developed with: CHCl
3
/CH
3
OH (9:1, vol/vol) as solvent I; CHCI
3
/CH
3
OH (8:2, vol/vol), as solvent II; or CHCI
3
/CH
3
OH/H
2
O (60:12:1, vol vol/vol), as solvent III. The sugar-containing compounds were visualized
by spraying plates with 2 % anthrone in concentrated H
2
SO
4
followed by heating at 110 °C. Acylated trehaloses appeared as anthrone-positive lipids
(blue spots) with a Rf value of 0.37 for DAT and a Rf value of 0.64 – 0.68 for TAT.
A final purification was carried in a TLC on silica gel-60 coated plates with a thickness
of 0.5 mm (E. Merck, Darmstadt, Germany). The lipids were recovered from the plates
and examined as mentioned before. The fractions with purified DAT were pooled, dried
and subjected to the Lymulus test to verify endotoxin contamination. A similar procedure
was followed to purify TAT.
Characterization of dat and tat by fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) analyses of the lipids were recorded
in a Vector 33 FTIR spectrometer (Bruker Corporation, Billerica, MA, USA), equipped
with an attenuated total reflection (ATR) module. About 0.5 mg of the product was
dissolved in 200 ?l chloroform-methanol (9:1, vol/vol) and placed into the ATR cell.
FTIR spectrum measurement was performed in wave number range of 4000-450 cm
?1
.
Assays to study the effects of dat and tat on nitric oxide production in macrophages
Six to seven week old Balb/c-J mice were used. To obtain MØs, bone marrow cells were
flushed from femurs and tibias and cultured in RPMI 1640 with 20 % FBS, supplemented
with 1 % non-essential aminoacids, 1 % of antibiotic-antimycotic and 1 % sodium pyruvate
(Invitrogen, Eugene, OR, USA). Cells were grown at 37 °C with 5% CO
2
. At day 10, MØs were obtained, and the cell viability was assessed with Trypan blue.
In preliminary experiments to determine the capacity of bone marrow-derived MØs from
Balb/c-J mice to produce NO, the cells were treated with various amounts of LPS (E.coli
B55:05; Sigma Chemical Co, St Louis, MO, USA) or recombinant IFN-? (BioLegend, San
Diego, CA, USA). The glycolipids were dissolved in hexane:ethanol (1:1, v/v). To each
well, 20?g glycolipid in 100 ?l hexane:ethanol were added and allowed to evaporate
to dryness; control wells received solvent alone. Then, 1?×?10
6
MØs were added to the wells and incubated for 24h at 37 °C with 5 % CO
2
. Afterward, 500 ?g LPS or 250ng IFN-? were added to the wells. Control wells with
only hexane:ethanol, DAT or TAT were included. After 24 h the isolated supernatants
were mixed with an equal volume of Griess reagent (1 % sulfanilamide, 0.1 % N-1-naphthylethylenediamine
dihydrochloride, and 2 % phosphoric acid) (Promega Co., Madison,WI, USA) and incubated
at room temperature for 10 min. Absorbance was measured at 550 nm.
Western blot and flow cytometry to determine the expression of iNOS in macrophages
treated with DAT and TAT
To investigate the expression of iNOS by MØs treated with the glycolipids, the cells
were lysed with RIPA buffer and the proteins were separated in 7.5 % PAGE-SDS gels,
transferred to a PVDF membranes and incubated overnight with a monoclonal antibody
diluted 1:100 to murine iNOS (BD Biosciences, San Diego, CA, USA). Membranes were
extensively rinsed with PBS and incubated with a secondary antibody to mice IgG diluted
1:200, 2 h at room temperature. The reactive bands were visualized by chemiluminescence
with SuperSignal West Dura kit (Pierce, Rockford, IL, USA) or with DAB/H
2
O
2
. For flow cytometry, 5?×?10
5
cells were fixed with paraformaldehyde 1 %, permeabilized with saponin 0.05 % and
incubated 1 h with the monoclonal antibody diluted 1:200 to murine iNOS. The cells
were rinsed and incubated for 1 h with a secondary antibody labeled with FITC. The
cells were analyzed in a Beckton Dickinson cytofluorometer (San Diego, CA, USA). As
a control, a monoclonal antibody of the same isotype was used.
Statistical analysis
Statistical analysis was performed using the standard statistical software Prism version
5.0, GraphPad Software, (San Diego, CA, USA). NO and iNOS production by cells was
expressed as inhibition percentages, where LPS or IFN-? induced levels, in the absence
of mycobacterial lipids, were taken as 100 %.