Identification of genes related to susceptibility to arthritis
Karina Toledo/Agência FAPESP/DICYT In experiments on mice, researchers at the Butantan Institute have identified a set of genes involved in susceptibility to rheumatoid arthritis – a chronic inflammatory and autoimmune disease that mainly affects the joints.
The findings were published in a February article of the journal PLOS One. If validated in humans, they will pave the way for the development of therapies and tests that will allow to predict disease progression.
“The identification of target genes offers several options for action. We can try to regulate their function with medication or through molecular genetic techniques, thus reducing the severity of the arthritis. The genes may also serve as markers of prognosis, guiding the treatment,” said Marcelo De Franco, Butantan researcher and coordinator of the FAPESP-funded project, “Identification of inflammatory process modulator genes using the combined analysis of genomic fine mapping and global gene expression.”
In individuals who are genetically predisposed to developing rheumatoid arthritis, the immune system attacks the synovial membranes – the thin layer of connective tissue that covers the joints (in the hands, wrists, elbows, knees, ankles, feet, shoulders, and spine) – as well as organs such as the lungs, heart and kidneys.
“No one knows exactly how the arthritis process is triggered, but we do know that some people are more susceptible to it than others. Genetic factors are what provide this predisposition, and we wanted to discover these factors by using this experimental model,” De Franco explained.
The experiment was conducted with two lines of mice that the Butantan Institute has used for nearly 20 years: one develops a minimal acute inflammatory reaction and has low susceptibility to arthritis (AIRmin), and the other develops a maximal acute inflammatory reaction and has higher susceptibility to the disease (AIRmax), De Franco explained.
“We used traditional methods of animal selection, the same as are used with livestock. Breeding was conducted, and in each generation, animals that had higher or lower inflammatory capacity were selected. In the end, we obtained two groups with all the genes responsible for the phenotypes of high or low inflammation in homozygosis, but with a heterogeneous genetic basis (like the human population). The model therefore offers advantages over isogenic mouse lines, which are all clones,” he said.
Over the course of the past two decades, several projects funded by FAPESP have contributed to the development of the AIRmax and AIRmin mouse lines, among them the thematic project “Identification of genetic factors affecting resistance and susceptibility to chemical carcinogenesis and the degree of acute inflammatory response, using a model of genetically selected mouse lines,” coordinated by Olga Celia Martinez Ibanez.
To map the genomic regions related to inflammation, the researchers bred the two lines of animals. Then, the mice from the first generation of offspring (F1) were crossbred, giving rise to 290 second-generation offspring (F2) mice.
“The genetic markers related to the inflammatory response and to susceptibility to arthritis have already been described in the scientific literature, but we wanted to prove that they were in fact associated with the phenotype of increased inflammation. Therefore, we bred the animals and went back to the initial condition of the selective process. We then investigated whether the animals in the F2 generation that had a higher inflammatory response presented the expected genetic markers,” De Franco explained.
The researcher went on to explain that these genetic markers may be a gene or a genomic sequence that has variations in a population and has a known chromosomal location. Examples of this are the genes that determine blood type.
“We used genomic sequences that included single nucleotide polymorphisms (SNPs), in other words, sequences that presented a variation in a single DNA base-pair. As in a paternity test, 1,500 markers were used. The marker has a specific polymorphism that may come from the father (high inflammation, for example) or from the mother (low inflammation). This allows us to identify chromosomal regions related to inflammation and to the development of arthritis,” De Franco explained.
To induce the inflammatory process in the mice, the researchers injected a suspension of polyacrylamide (a type of polymer) microparticles into the dorsal region of the animals. After a 24-hour period, a saline solution was injected into the same area, and the inflammatory infiltrate was collected to count the number of ‘defense’ cells, mostly neutrophils.
“To induce arthritis, we injected a mineral oil called pristane into the peritoneum of the animals. Nearly 90 days later, they developed the pathology [arthritis], which was measured by the extent of swelling of the paws, the number of autoantibodies, the expression of inflammatory cytokines and the histology of the paws. The susceptible mice had swollen paws, autoantibodies and high expression of inflammatory cytokines, whereas the resistant mice did not,” said De Franco.
After mapping the chromosomal regions related to susceptibility or resistance to arthritis, the researchers went back to the initial lines – AIRmin and AIRmax – to analyze the gene expression from these locations.
“We looked in these chromosomal regions for genes with differentiated expression in the two lines. We found some candidates and then conducted experiments to modify the expression of these genes to verify whether the phenotype would then be altered,” De Franco explained.
Among the candidate genes found were Slc11a1 on chromosome 1, Pycard on chromosome 7, and Cxcl1, Cxcl9, Cxcl5 and Cxcl13 on chromosome 5.
“Slc11a1 is one of the most effective genes because it regulates the action of macrophages – cells that are very important in modulating the inflammatory response. Thus, Slc11a1 end up regulating the expression of a series of other genes related to inflammation. When the expression of this gene was modified, there was not only a change in the phenotype, but also in the expression of other genes related to arthritis and inflammation,” De Franco explained.
To modify the expression of the candidate genes, the researchers used a technique known as “knockout false.” Mice with the defective gene were cross-bred to make them into homozygotes, De Franco explained.
“In this way, we obtained mice of the high inflammation line AIRmax that had the defective Slc11a1 gene and another AIRmax line that carried the normal gene. We did the same for the animals with weak inflammation, the AIRmin mice. The breeding was helped by genotyping because, before breeding, we identified whether the gene was normal or defective using molecular biology methods, such as the polymerase chain reaction (PCR),” said De Franco.
According to the researcher, although humans and mice have different numbers of chromosomes, it is already known which chromosomal regions correspond between the species, which allows the establishment of parallels.
“The next step is to further investigate the interaction of these genes in order to discover exactly how they regulate the inflammatory response and to begin to validate the findings in human models,” De Franco said.