Synthesis of multivalent tuberculosis and leishmania-associated capping carbohydrates reveals structure-dependent responses allowing immune evasion

Article abstract of DOI:10.1021/ja103351m

Mycobacterium tuberculosis and the protozoan parasites of the genus Leishmania are intracellular pathogens that can survive in macrophages-the very white blood cells of the immune system responsible for engulfing and ultimately clearing foreign invaders. The ability of these pathogens to hide within immune cells has made the design of effective therapies, including vaccines, to control tuberculosis and leishmaniasis particularly challenging. Herein we present the synthesis and development of carbohydrate-based probes to demonstrate that changes in pathogen-associated surface oligosaccharides are sufficient to alter cellular immune responses and thereby let a pathogen hide from immune surveillance.

Full text of DOI:10.1021/ja103351m

Published on Web 07/29/2010
Synthesis of Multivalent Tuberculosis and Leishmania-Associated Capping
Carbohydrates Reveals Structure-Dependent Responses Allowing Immune
Evasion
Eun-Ho Song,† Alex O. Osanya,‡ Christine A. Petersen,‡ and Nicola L. B. Pohl*,†
Department of Chemistry and The Plant Science Institute, Gilman Hall, Iowa State UniVersity, Ames, Iowa
50011-3111 and Department of Veterinary Pathology, Veterinary Medicine, Iowa State UniVersity,
Ames, Iowa 50011-1250
Received April 20, 2010; E-mail: npohl@iastate.edu
various immune responses,7 herein we present the first demonstra-
Abstract: Mycobacterium tuberculosis and the protozoan para-
tion that a synthesized trimannose cap sugar of ManLAM alone is
sites of the genus Leishmania are intracellular pathogens that
can survive in macrophagessthe very white blood cells of the
immune system responsible for engulfing and ultimately clearing
foreign invaders. The ability of these pathogens to hide within
immune cells has made the design of effective therapies, including
vaccines, to control tuberculosis and leishmaniasis particularly
challenging. Herein we present the synthesis and development
of carbohydrate-based probes to demonstrate that changes in
able to alter IL-12 production of macrophages via a Toll-like
receptor (TLR)-2 dependent manner. We present the synthesis and
development of new chemical tools to demonstrate that different
surface carbohydrates are sufficient to change cellular immune
responses and thereby let a pathogen hide from immune surveillance.
One of the most abundant molecules found on the cell surfaces
of bacteria, parasites, and viruses are carbohydrates.8 Therefore,
we reasoned that carbohydrates could be at least partially respon-
sible for differences in immune responses seen between virulent
and nonvirulent pathogens. We envisioned making “artificial
pathogens” of approximately the same size (one micrometer) as
pathogen-associated surface oligosaccharides are sufficient to
alter cellular immune responses and thereby let a pathogen hide
from immune surveillance.
Leishmania and M. tuberculosis that are coated only with the well-
defined carbohydrate capping structure associated with these
pathogens to compare the difference in cellular response when only
the carbohydrate coat is altered. Such clean comparison experiments
are impossible to do through genetic modification of the carbohy-
drate coat of the pathogens themselves because the gene products
responsible for synthesis of the capped structures also have other
functions.9
Mycobacterium tuberculosis and the protozoan parasites of the
genus Leishmania are intracellular pathogens that can survive in
macrophagessthe very white blood cells of the immune system
responsible for engulfing and ultimately clearing foreign invaders.
The ability of these pathogens to hide within immune cells has
made the design of effective therapies, including vaccines, to control
tuberculosis and leishmaniasis particularly challenging. Tuberculosis
claims two million deaths annually worldwide;1 leishmaniasis is
endemic over much of 88 countries in Africa, India, southern
Europe, and Central and South America.2 Mycobacterial glycolipid
mannosylated lipoarabinomannan (ManLAM, Figure 1) is a major
contributor to M. tuberculosis evasion and subversion of host
immune responses.3 All pathogenic strains of Mycobacterium (M.
tuberculosis, M. leprae), as well as the vaccine strain BCG, bear
caps consisting of either a dimannoside or a trimannoside. This
mannose cap is a major structural element engaged in receptor
binding due to its prominent external location and is important in
determining immunopathogenesis of Mycobacteria spp.3 The cy-
tokine IL-12 is critical for production of a productive T helper type
1 immune response against either M. tuberculosis or Leishmania
spp. Treatment of cells with ManLAM from either M. tuberculosis
or BCG results in very limited IL-12 production leading to a
nonpolarized, mild immune response.4 ManLAM IL-12 inhibition
was found to correlate with the presence of mannose caps or
glycosylphosphatidylinositol (GPI) acyl residues.4b Oligotriose and
dipalmitoylphosphatidylethanolamine (Man 3-DPPE) treatment was
found to induce IL-12 production in peritoneal macrophages.5
Although many groups have documented the role of the lipid portion
of these glycolipids in pathogen virulence6 and enzymatic cleavage
of various mannan and glucan structures have been shown to alter
Figure 1. Capping structure in lipoarabinomannan (ManLAM) of M.
tuberculosis and lipophosphoglycan (LPG) of Leishmania.
Alpha-linked trimannose is one of the major components in both
the outer lipophosphoglycan (LPG) of Leishmania parasites10 and
the lipoarabinomannan (LAM) of M. tuberculosis cell walls11 and
therefore makes an excellent synthetic target (Figure 1). To date,
several synthetic approaches have been reported to construct
Leishmania capping structures including oligomannose and branched
oligosaccharides primarily for vaccine development.12 We required
a route to the trimannose structure that would allow a handle at
the reducing end of the sugar to attach it to a polymeric bead for
a multivalent display. The known trimannoside 212c was then
prepared using fluorous solid-phase extraction (FSPE)-based itera-
tive synthesis from mannose trichloroacetimidate 1 (Figure 1). The
† Department of Chemistry and The Plant Science Institute.
‡ Department of Veterinary Pathology.
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C O M M U N I C A T I O N S
fluorous-tag was cleaved by ozonolysis; oxidation with Jones
reagent provided a carboxylate handle for further coupling with
amine-functionalized beads. Global deprotection with sodium in
ammonia at -78 °C furnished the desired fully deprotected
trimannose 4 in good yield. As a control, lactose was prepared with
the same linker by a similar sequence from the known allyl
lactososide.13
With the two desired beads in hand, we examined the effect of
the sugar-coated beads on the inflammatory response mediated by
macrophages. Murine macrophages were first stimulated by the
known activational molecules, lipopolysaccharide (LPS) and in-
terferon gamma (IFNγ). Lactose-coated beads B1 and trimannose-
coated beads B2 were incubated with the stimulated macrophages,
and the levels of the proinflammatory cytokine interleukin-12 (IL-
12) produced were measured 24 h postexposure.20 IL-12 plays a
significant role in the link between innate and adaptive immunity21
(Figure 2). Comparable levels of IL-12-p40, the regulated portion
of the bioactive IL-12p70, were produced when macrophages were
exposed to lactose-coated beads B1 and the sugar-free beads B0.
In contrast, the presence of the trimannose-coated beads B2
significantly diminished IL-12p40 production. The innate immune
response was significantly dampened to normally stimulatory signals
only by the change of the carbohydrate structure presented on the
bead.
To test for the possible mechanism of this dampening effect,
the macrophages were treated with antibodies against the extra-
cellular lectin binding domain of Toll-like receptor 2 (TLR2). TLR2
is known to play a crucial role in pathogen recognition and
activation of innate immune responses.22 Blocking TLR2 eliminated
the differences in production of IL-12p40; there was no difference
between the trimannose-coated or negative control coated beads.
Isotype IgG1 antibodies were used as a control to demonstrate that
the effect was specific to antibodies against TLR2. The possibility
of recognition via mannose receptors4b was also investigated via
antibody inhibition and was not found to have a significant effect
in leading to alteration of IL-12p40 production (data not shown).
These data clearly implicate a TLR2-mediated pathway for this
newly discovered trimannose-mediated immune suppression.
With the capping sugar and the control sugar lactose in hand, a
suitable 1 µm sized support was required for multivalent display
of these sugars. Latex beads are commonly used in immunoassays
due to their inertness and commercial availability. Moreover, beads
with high concentrations of imbedded fluorophores enable various
fluorescent assays.14 For example, such beads have been utilized
for agglutination tests for the detection of antibodies or investigation
of heparin-binding properties, for the analysis of blood cell
populations, and for the identification of specific cell membrane
markers.15 Commercially available latex beads B0 (microspheres,
1 µm diameter and yellow-green fluorescent) derivatized with
carboxylate groups (3.5 × 10-4 mmol carboxylate groups per mL)
were chosen because the size of these beads mimics the size of the
Leishmania and M. tuberculosis pathogens and the FITC-label
allows use of a common immunofluorescent assay for the observa-
tion of bead uptake by macrophages. In order to avoid the spatial
proximity of sugars on the surface of the beads and improve the
accessibility of sugars to possible macrophage binding partners,
an ethylenediamine spacer was attached to the carboxylated bead
surface under standard peptide coupling conditions (Scheme 1).
Although numerous coupling reagents,16 including carbodiimides,
phosphonium-based reagents, and aluminum-based reagents, have been
utilized for amine coupling reactions, restrictions such as stability of
generated intermediates, byproduct, and cost determine the choice of
reagent for solid-phase synthesis under aqueous conditions. Among
the relatively inexpensive carbodiimide coupling reagents, 1-ethyl-3-
[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) mediated
coupling reactions with N-hydroxysuccinimide (NHS)17 have the
advantage of formation of a relatively stable NHS-ester intermediate
and also allow easy purification to eliminate urea byproduct or excess
EDC/NHS. A combination of EDC and NHS in deionized water
proved successful for the synthesis of the necessary amide linkages
(Scheme 1). The subsequent coupling reaction of carboxymethyl-
trimannose and -lactose with a bead-linked amine spacer gave the
desired beads B1 and B2. Each coupling step was repeated twice with
30 to 50 equiv of the sugar to ensure complete reaction. The density
of sugar was calculated by the given density of the carboxylates (1.296
× 10-14 mmol/bead). In addition, the Kaiser colorimetric test was used
as an indicator of coupling reaction progress.18 Finally, a phenol-
sulfuric acid assay was also used to confirm sugar loading densities
on the bead surfaces.19
Scheme 1. Synthesis of Multivalent Tuberculosis and Leishmania
Capping Structures on Latex Beads
Figure 2. Effect of cap sugar-coated latex beads (B0 ) latex bead, B1 )
lactose coated beads, and B2 ) trimannose-coated beads) on the production
of IL-12p40 by murine macrophages, J774 cells, stimulated by LPS and
IFNγ. [Asterisks denote a significant change between the TLR2 blocking
from control isotype and/or nonblocking-stimulation only treated cells (p
< 0.05) via Student’s t-test.] For comparison, no stimulation resulted in
200 ( 50 ng/mL IL-12p40 production. Stimulation alone had the same
effect as stimulation with the latex bead (B0).
In conclusion, the synthesis of pathogen mimics with well-defined
carbohydrate coats allowed the first demonstration that carbohy-
drates alone in the appropriate context are sufficient to dampen
innate immune responses mediated by macrophages via TLR2-
dependent pathways. The revelation that these carbohydrate moieties
produce immune response alteration is critical to the production of
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Acknowledgment. This work was supported in part by a
Healthy Livestock Initiative Competitive Grant from Iowa State
University and a grant from the NIH (1 R21 AI074711-01A2). We
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Supporting Information Available: Synthetic protocols, assay
conditions, and NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
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