10 April, 2009

No Dendritic Cells --> Unchecked CD4+ T-Cell Proliferation --> Autoimmunity --> DEATH

ResearchBlogging.orgDendritic cells (DCs) are the extremely important interface cell type between the innate and adaptive immune systems. Without them, the adaptive immune system has a very hard time getting started and the organism has a much more difficult time fighting off pathogens. But DCs don't just save our lives when we're sick, they also save us from ourselves when we're not. It has recently been found that constitutive knockout of dendritic cells leads to the development of spontaneous fatal autoimmunity.

[Context/background in previous posts on dendritic cells, T-cells, asthma T-cells, and multiple sclerosis autoimmunity.]

As is the case in many immune system pathologies, this comes back to T-cells. In a normal thymus, T-cells are continually proliferating in huge numbers. Each T-cell undergoes Rag-mediated receptor specificity recombination and randomization while it is forming its receptor. This gives rise to T-cells with receptors specific to a massively diverse array of potential antigens and is at the core of how the adaptive immune system recognizes pathogen peptides and fights back against them specifically. However, most of the T-cells that get made are killed off summarily for having receptors that react against self-antigens (meaning peptides native to the organism; called autoreactivity). Although the thymic epithelium is also involved in screening potential T-cells for autoreactivity, DCs are the primary mechanism by which these potentially harmful T-cells are weeded out before they can escape from the thymus differentiated and initiate an autoimmune response. There is even a specialized subset of thymic DCs that express diverse self-antigens to help weed the T-cells reacting against them out, and systemic plasmacytoid and myeloid DCs often circulate through the thymus or lymph nodes bearing chunks of apoptotic self-cells.

This group found a way to constitutively delete DCs. Without the DCs, the CD4+ T-cells of the mice went wild and proliferated massively because they weren't being tested and usually killed anymore (~10-fold more IFNg- and IL-17A-producing CD4+ T-cells in DC- than DC+ mice). This allowed self-reactive T-cells to mature and get out into the circulation, where they infiltrated various tissues and caused massive inflammation. Many of the mice used here died after the autoimmune response had developed. It should be noted that the DC deletion wasn't absolute, IFN-producing DCs were unaffected by their construct and B-cells and some macrophages have been shown to be able to prime T-cells as well, which explains how the CD4+ T-cells were able to fully mature in DC- mice.

Not only did effector inflammatory CD4+ T-cells increase, but Treg cells increased in frequency also, they just didn't seem to be doing much. They seemed to see that effector inflammatory CD4+ T-cells were out of control and tried to proliferate to help curb this, but without DCs they couldn't actually make anything happen. This prompted a quick preliminary search through PubMed and Google Scholar and it looks like the IL-10 that gets produced by Tregs decreases DC maturation from monocytes and encourages macrophage formation instead. It also looks like immature pre-DCs exposed to IL-10 don't express as much CD4/8 or MHCI/II as fully differentiated DCs. This is interesting in that is strongly suggests Tregs don't act directly on inflammatory T-cells, but instead act through DC-mediated pathways.

The group also infected DC- mice with helminths to provoke an immune response and see what happens. Not only were DC- mice unable to clear the infection as DC+ mice were, but DC- also had reduced pulmonary eosinophilia and increased TH1 and TH17 type effector T-cell tissue infiltration (oddly, they didn't report on any TH2 type data, which would have been very interesting). This demonstrated that DCs were absolutely required to mount an effective immune response to infection, which apparently hadn't been definitively shown before (probably because it's hard to knock DCs out; see below).

However, even though DC- mice weren't able to clear a helminth infection, they were able to make antibodies. This defies classical immunology dogma because it is widely supported that T-cells go and activate B-cells to make antibodies after getting primed by DCs. Under some circumstances, e.g., this DC- one, T-cells can still get primed by B-cells or peripheral monocyte populations, which may include the IFN-producing DCs that didn't get knocked out.

To test for antibodies from DC- mice, they used tissue sections from rag-/- mice and immunofluorescently stained them for mouse IgG. Rag is the gene that controls the formation of unique T- and B-cell receptors, and without the rag gene the organism has absolutely no adaptive immune response* and makes no antibodies whatsoever, although they still do have innate immune effector cells. Therefore rag-/- mouse tissues will have no antibodies and no background staining, allowing detection of autoreactive antibodies from the DC- mice. The immunofluorescence staining showed that the targets of autoimmunity varied widely, with some mice autoreactive to nuclear components, others to lamina propria components, and still others to the epithelium itself. Why is this important? Antibodies are the adaptive immune system's signal to the innate immune system that something the innate immune system otherwise wouldn't see as bad is, in fact, bad and should be destroyed. So if something gets antibody bound to it, the innate immune effector cells (i.e., neutrophils, macrophages, eosinophils, even DCs) will recognize it as bad and chew on it. In the case of the DC- mice, the unregulated adaptive immune system wound up labeling the mouse's own tissue as bad and initiated inflammatory responses against it, sort of shooting itself in the foot.

In summary, DCs are important not only to fight off infection, but also to keep the adaptive inflammatory responses in check. Dysregulation or maldevelopment of DCs is therefore a potential target for remediation in autoimmune conditions such as inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis.

Genetics Addendum: The DC-knockout construct that this group made was really elegant and cool. I didn't mention it above because I wanted to stick to the cool big stuff, but this here is some awesome little stuff. Cre is a recombinase that recognizes floxP sequences. When there are 2 floxP sequences flanking a gene and when Cre gets expressed, it recognizes those floxP sequences and cuts out everything between. This system underpins 17 metric craploads of transgenic mouse studies.

Here, they built a construct where Cre was constitutively (this means always) expressed by a DC-specific promoter. They also stuck in a 3-layer sequence elsewhere on the construct. The outer layer of the sequence consisted of 2 complementary chunks of the sequence for diptheria toxin, the thin middle layer consisted of the floxP sequences, and the inner layer was an erythropoetin resistance gene. So without the Cre, the erythropoetin resistance gene disrupted the diptheria toxin gene. But when Cre got expressed in developing DCs in the bone marrow, the floxP sequences and the erythropoetin resistance gene got cut out and joined the diptheria toxin back into a functional sequence. So then diptheria toxin got made and killed the developing DCs before they could even get out of the bone marrow.

*This makes rag-/- mice popular for studying T-cell behavior. In these experiments, T-cells are taken from a rag+/+ mouse, manipulated according to experimental aims, and injected into rag-/- mice where their behavior and dynamics may be readily observed without native interference.

Ohnmacht, C., Pullner, A., King, S., Drexler, I., Meier, S., Brocker, T., & Voehringer, D. (2009). Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity Journal of Experimental Medicine, 206 (3), 549-559 DOI: 10.1084/jem.20082394

1 comment:

Contract Research said...

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Thank you very much for your ideas to post comments. Dendritic cells are antigen-presenting cells, which play a critical role in the regulation of the adaptive immune response. These signaling cascades lead to the expression of various cytokine genes...