David Segal

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.

Giorgio Trinchieri

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
At TLR3

TLRs are a class of germ-line–encoded 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.

Jessica Bell

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.

Felix Yarovinsky

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.

Brian Kelsall

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 10–14 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. 

Daniela Verthelyi

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