Infection-fighting protein could be key to autoimmune disease, say U-M scientists

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"IL-1beta is a master regulator of infection, and it's known to be involved in the development of rheumatoid arthritis," says Gabriel Nunez, M.D., a professor of pathology in the U-M Medical School, who directed the research study. "So it's likely that these findings will apply to other autoimmune diseases, as well."

In a study being published Jan. 11 as an Advance Online Publication in Nature, U-M scientists show, for the first time, that cryopyrin is activated by bacterial RNA and that it is essential to the cell's ability to mount an effective defense against bacteria.

Found in the cytosol, or fluid inside cells, cryopyrin is a member of the NOD-LRR family of proteins, which protect cells against microbial infection. Defective cryopyrin is predicted to be associated with increased susceptibility to infection.

Small mutations in CIAS1 - the human gene for cryopyrin - are known to cause three rare autoinflammatory diseases: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease. People with these diseases produce uncontrolled amounts of IL-1beta and other inflammatory molecules. This causes them to have recurrent episodes of fever and to develop rashes - often when they are exposed to cold temperatures.

Based on previous research with cell lines, scientists suspected that cryopyrin was an important link between the immune system's normal job of killing bacteria and the abnormal development of autoimmune diseases. But no one was sure exactly how cryopyrin was "turned on" in living animals or how it stimulated the immune response.

In previous research, the U-M team found that the single-point mutation in CIAS1 - which causes autoinflammatory syndromes in people - activates cryopyrin, even when there is no bacterial RNA present in the cell. "The mutation fools the cell into producing the activated form of cryopyrin, even when bacteria aren't there," Nunez says.

To decipher cryopyrin's signal, Thirumala-Devi Kanneganti, Ph.D., a U-M post-doctoral research fellow in pathology, studied immune cells called macrophages and several strains of laboratory mice. One of these strains was unable to produce cryopyrin, because the CIAS1 gene had been removed.

Kanneganti exposed the macrophages and mice to bacterial RNA and to small synthetic molecules called R837 (Imiquimod) and R848 (Resiquimod). These adjuvant molecules activate the pro-inflammatory response in mice and are used as anti-tumor agents and to treat genital warts caused by a virus in human patients.

"We found that cryopyrin was activated and the macrophages began secreting IL-1beta following stimulation with R837 or R848," Kanneganti says. "Since the structure of these molecules is very similar to DNA or RNA, we believe the natural ligand, or activating molecule, for cryopyrin could be DNA or RNA."

In previous research, other scientists discovered a signaling pathway in which molecules called toll-like receptors on the cell's surface recognize invading bacteria and activate the immune response. But U-M scientists found that cryopyrin uses a different signaling pathway. Activated cryopyrin triggers an enzyme called caspase-1, which splits the immature form of IL-1beta to produce the active form of the molecule. Once IL-1beta is activated, it can be secreted out of the cell where it binds to the IL-1beta receptor on other cells to trigger an immune response.

"These two signaling pathways cooperate," Nunez explains. "The toll-like receptor pathway recognizes bacteria outside the cell, while cryopyrin recognizes bacteria that's already in the cell. When a toll receptor on the membrane senses bacterial RNA, it activates a signaling pathway called NF-kappaB, which induces the production of IL-1beta. Cryopyrin does the same thing, but it works through caspase-1 to produce the active form of IL-1beta."

In her experiments, Kanneganti confirmed that the signaling pathway requires the presence of cryopyrin. Macrophages and mice that lacked the CIAS1 gene for cryopyrin were unable to generate an immune response when exposed to bacterial products.

The research was funded by the National Institute of Allergy and Infectious Diseases (NIAID). The University of Michigan has filed a patent application on this research technology.

Additional U-M collaborators on the study included Nesrin Ozoren, Mathilde Body-Malapel, Amal Amer, Jong-Hwan Park, Luigi Franchi and Joel Whitfield. Other collaborators were Winfried Barchet and Marco Colonna from the Washington University School of Medicine, Peter Vandenabeele from Belgium's Ghent University, John Bertin, Anthony Coyle and Ethan P. Grant from Millennium Pharmaceuticals and Shizuo Akira from Japan's Osaka University.

Citation: Nature DOI: 10.1038/nature04517

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