Study helps understand how defense cells combat bacteria

Researchers find that different types of inflammasomes – protein complexes responsible for initiating an inflammatory response in macrophages – can regulate each other to amplify signals of infection.

By Karina Toledo  |  Agência FAPESP – When the defense cells that patrol the human organism come across a potentially dangerous bacterium, intracellular protein complexes known as inflammasomes are activated.

This mechanism is essential to trigger an inflammatory process that attracts a veritable army of immune cells to the site of the confrontation and halts the progress of the infection.

Elucidating exactly how such defense mechanisms work is the goal of a Thematic Project supported by FAPESP, with Dario Simões Zamboni as the principal investigator. Zamboni is a professor at the University of São Paulo’s Ribeirão Preto School of Medicine (FMRP-USP).

Recent results were published in July in the journal Cell Reports. The study is being conducted under the aegis of the Center for Research on Inflammatory Diseases (CRID), one of FAPESP’s Research, Innovation and Dissemination Centers (RIDCs).

“This kind of basic knowledge can be of help in the future development of new methods both to combat infections and to prevent the occurrence of exacerbated inflammation that damages the organism, as in the case of sepsis,” Zamboni told Agência FAPESP.

In their latest work, the group investigated the interaction among three different types of inflammasomes that can be activated in macrophages, which are front-line cells in the immune system that are responsible for phagocytizing (enveloping and destroying) potential invaders. 

One of these inflammasomes is triggered when certain molecules – which may or may not be microbial components – perforate macrophage membranes and allow potassium to leak out of the intracellular medium. The result is activation of a protein known as NLRP3.

The second type of inflammasome is activated when defense cells detect the presence of DNA from invading microbes in their cytoplasm, causing activation of the protein AIM2.

The third type is induced by the presence of LPS in the macrophage cytoplasm. Short for bacterial lipopolysaccharide, LPS is the major component of the outer membrane of Gram-negative bacteria, which include several bacteria that cause disease in humans, such as Escherichia coli, Shigella, Salmonella, Pseudomonas, and Legionella pneumophila. In this third case, the inflammatory process begins with activation of the protein caspase-11.

Albeit with activation mediated by different proteins, all three inflammasomes lead to the production of pro-inflammatory molecules such as interleukin-1 beta (IL-1β) and interleukin-18 (IL-18). These cytokines tell the immune system that its army, consisting of neutrophils, inflammatory monocytes, lymphocytes and other kinds of leukocytes, must be sent to the site. This process produces the typical symptoms of inflammation, such as pain, heat, redness and swelling. 

“The idea that these inflammasomes act independently of each other has predominated until now, but we’re showing that one of them can regulate activation of another so as to magnify the signs of infection and induce a more powerful inflammatory response,” Zamboni said.

Methodology

The experiments that made this conclusion possible were performed in vitro with cultured macrophages from mice and in vivo with infected murine lungs. The model involved infection with Legionella pneumophila, the bacterium that causes pneumonia and can activate multiple types of inflammasomes in macrophages.

“We observed in vitro that when the amount of bacterial DNA inside each defense cell is very small, the stimulus is not sufficient for the protein AIM2 to cleave the peptide bonds of another protein called caspase-1 and thereby activate the AIM2 inflammasome,” Zamboni said. “However, AIM2 is able to leave caspase-1 in its active form, and the two proteins work together to perforate the macrophage membrane, activating the NLRP3 inflammasome.”

According to Zamboni, previous research suggested that when the protein caspase-11 recognizes LPS in the macrophage cytoplasm, it also induces membrane damage that results in activation of the NLRP3 inflammasome.

“We’ve now shown that the AIM2 inflammasome also amplifies infection signals and activates the NLRP3 inflammasome in an unconventional way,” he added.

When they repeated the experiment in cells that had been modified so as not to express AIM2 and caspase-11, the researchers found that the NLRP3 inflammasome was not activated in the presence of the bacterium in the culture medium and that the cell became completely incapable of orchestrating an inflammatory response.

“When one of the two pathways, AIM2 or caspase-11, was allowed to remain working, the response occurred, but less efficiently than in the wild cell,” Zamboni said.

Next, experiments were performed with mice to see whether a similar effect would be observed in vivo, given that several immune factors interact in this situation.

Two groups were studied, with one comprising wild mice (without genetic modification) and the other comprising mice that had been modified so as not to produce AIM2 and caspase-11.

“When the wild mice inhaled the bacteria, all inflammasome pathways were activated, an effective response was orchestrated, and the infection was controlled,” Zamboni said. “In AIM2- and caspase-11-knockout mice, the bacteria replicated five to ten times more in the lungs during the first two days of infection.”

For Zamboni, there is evidence that during evolution, the mammalian immune system developed multiple mechanisms to recognize different elements of pathogenic microbes and activate the NLRP3 inflammasome, guaranteeing the production of an inflammatory response.

“Recognition of a microbe with the potential to cause damage is the first and one of the most important processes that defense cells have to perform,” he said. “We’re in contact with thousands of microorganisms all the time, and most of them don’t present a threat. Mounting an inflammatory response against pathogenic microbes is important but costly for the organism. It has to be worthwhile.”

The article “AIM2 engages active but unprocessed caspase-1 to induce noncanonical activation of the NLRP3 inflammasome,” which was featured on the cover of the July 2017 issue of Cell Reports, can be retrieved from cell.com/cell-reports/abstract/S2211-1247(17)30934-8.