T H E N I H C A T A L Y S T | N O V E M B E R D E C E M B E R 2005 |
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Research
Festival
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by Aarthi Ashok |
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David Segal
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Much
has been learned in recent years about the receptors of the innate immune system
the most important of which are the toll-like receptors (TLR)
and about dendritic cell function in host defense, observed David Segal, chief
of the Immune Targeting Section, NCI, and co-chair of the panel on innate immune
recognition. And what much of the new knowledge points to, he said, introducing
the talks to follow, is that "most cells of the body can serve some sort
of innate immune function."
The
Dendritic Cell Bridge
Dendritic cells come into play in the early stages of infection,
prior to activation of the adaptive immune system. Two different populations
of dendritic cells have been described: the conventional dendritic cells that
secrete primarily interleukin-12 (IL-12) and the plasmacytoid precursor dendritic
cells (pDCs) that secrete mostly type 1 interferon (IFN- a
or -b).
Giorgio
Trinchieri, a Fogarty Visiting Scholar at NIAID, and his colleagues were
interested in determining whether pDCs were the primary source of type 1 IFN
during infection in vivo. In 2001, they characterized the mouse counterpart
of human pDCs. Using specific antibodies in vivo to deplete this murine pDC
population, they were able to establish that mice subjected to microbial challenge
could no longer synthesize type 1 IFN.
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Giorgio Trinchieri
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Moreover, they were able to show that this inhibition of
type 1 IFN production was pathogen specific. pDCs express high levels of TLR7
and 9 but lack TLR3 and 4. Pathogens recognized
by TLR9 trigger a large type 1 IFN response, which is completely inhibited upon
antibody depletion of pDCs. Type 1 IFN production persists, however, in the
face of infection with pathogens that signal through TLR3, or a cytoplasmic
double-stranded RNA receptor such as RIG-I, despite antibody-mediated depletion
of pDCs. Their data, Trinchieri said, point to a role for the classical dendritic
cells in type 1 IFN production during later stages of viral infection.
Optimal production of the pro-inflammatory cytokine IL-12
during infection requires type 1 IFN. Trinchieri showed that pathogen stimulation
of multiple TLRs, which triggers classical dendritic cells, would resulted in
optimal IL-12 production.
A
Closer Look
TLRs are a class of germ-lineencoded pattern recognition
receptors that are truly our first line of defense against pathogens, said Jessica
Bell, a postdoc in the Laboratory
of Molecular Biology, NIDDK.
In humans, 10 members of this family have been described
to date (TLR1 to 10). Bell and co-workers probed the molecular structure of
TLRs in an attempt to ascertain how so few could recognize so immense a range
of pathogens and foreign molecules.
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Jessica Bell
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The TLRs contain leucine-rich repeat (LRR) domains that,
unlike other LRR proteins, contain insertions in specific positions. Bell suspected
that these insertions might be involved in antigen recognition.
Using a baculovirus secretion system and affinity chromatography,
Bell was able to produce the apo crystals that led to the determination of the
first structure of TLR3 a curved solenoid that resembles a Slinky toy
with an extended b-sheet structure on its concave
surface.
The structure contained 11 glycosylation sites out of a
potential 15, as well as two bound sulfate ions. When a 19-base pair RNA molecule
is modeled onto the receptor using the sulfate ions as landmarks for the nucleotide's
phosphate backbone, Bell noted, the glycosylation sites may direct the target
to the binding site.
Generation of co-crystals of TLR3 and double-stranded RNA
are underway, and Bell hopes to embark on a mutational analysis of the predicted
RNA binding sites on TLR3.
Innate
Immunity
To
Protozoan Parasites
Innate immune recognition of eukaryotic pathogens is little
understood. Felix
Yarovinsky and his colleagues in the Laboratory
of Parasitic Diseases, NIAID, have shown that MyD88-knockout mice, which
lack the downstream signaling from TLRs, fail to produce the IL-12 necessary
to combat the parasite Toxoplasma gondii. This finding was the first
that pointed to a role for TLRs in the recognition of and defense against parasitic
pathogens.
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Felix Yarovinsky
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The team then fractionated parasite extracts to identify
a single protein a 17.5-kDa novel profilin that was the actual
trigger for cytokine production from dendritic cells and hence a potential TLR
ligand.
Using cells from various TLR-knockout mice, Yarovinsky demonstrated
that the parasitic profilin protein is recognized by TLR11. Interestingly, profilins
from several other protozoans, including Cryptosporidium parvum and the
malarial parasite Plasmodium falciparum, are recognized by TLR11. Hence,
TLR11 appears to have evolved to recognize so-called apicomplexan parasites,
and innate responses are clearly critical in the clearance of these parasites.
TLR11-knockout mice can resist the acute phase of parasitic
infection but succumb to the chronic phase. Yarovinsky is now trying to identify
the source of the residual IL-12 that helps these mice combat the acute infection.
Innate
Immunity
To
Intestinal Reovirus Infection
Brian
Kelsall, a senior investigator in the Laboratory of Clinical Investigation,
NIAID, and his co-workers have been exploring the role of mucosal immunity during
infection with the double-stranded RNA-containing reovirus.
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Brian Kelsall
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Reovirus infects the epithelium overlying the Peyer's patches
in the gut and is cleared by a combination of innate and adaptive immunity in
about 1014 days.
Dendritic cells (DC) are prominent in Peyer's patches. Kelsall
found that while reovirus does not actively infect DCs, DCs can capture
reovirus structural proteins from infected apoptotic epithelial cells and present
them to CD4+ T cells in vitro. In addition, they demonstrated that type 1 IFN
is produced in Peyer s patches upon reovirus infection and is critical for reovirus clearance.
Reovirus did not induce type 1 IFN production from pDCs
in the Peyer's patch; interestingly, Kelsall said, the investigators found that
Peyer's patch pDCs did not make type 1 IFN in response to any stimuli, perhaps
due to conditioning by factors present in the mucosal microenvironment.
They concluded that type 1 IFN production by a non-pDC source
in the Peyer's patch is critical for early immunity. This type 1 IFN may also
be involved in driving DC activation, resulting in acquired immune responses
to this model intestinal virus infection.
Innate
Immune Activation
Studies have shown that innate immune activation using CpG
motifs (DNA sequences from bacterial molecules) protects mice against several
pathogens, especially hemorrhagic pathogens.
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Daniela Verthelyi
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Daniela
Verthelyi, a senior staff fellow in the Center for Drug Evaluation and Research,
FDA, and her colleagues have evaluated the immunoprotective effects of one family
of CpG oligonucleotides (D series) in rhesus macaques challenged with the parasite
Leishmania major. Their data show that the D-series oligos can confer
protection, inducing resolution of disease lesions within a few days of treatment.
Moreover, these immunoprotective effects are evident in SIV-infected immunocompromised
macaques.
There is a catch, however. The tails of the D-series oligos have a tendency to polymerize and aggregate in solution, changing the characteristics of the product. To navigate around this roadblock, the team first had to create D-series oligos with additional protective groups on the poly G tail that allowed them to become activated only upon entry into cells. These novel D-series oligos showed significant protective effects against Leishmania infection in macaques, albeit with some induction of local inflammation. n