Altered Signaling in Immune Cells May Contribute to Sjögren’s

Immune macrophages — immune cells that fight infection — can mount distinct responses to specific pathogens, based on how a single signaling molecule behaves over time in the presence of that stimuli, a recent study found.

However, macrophages in a Sjögren’s syndrome mouse model lack some of these immune signaling dynamics, making them less accurate at distinguishing between different microbes. This may trigger the onset of the autoimmune condition, the scientists said. 

The study, “Six distinct NFκB signaling codons convey discrete information to distinguish stimuli and enable appropriate macrophage responses,” was published in the journal Immunity. 

Sjögren’s syndrome is a chronic autoimmune disorder in which a misguided immune response targets the glands that produce saliva and tears. Studies suggest genetic changes within immune cells and those associated with inflammatory pathways may trigger the altered immune response.

Macrophages are immune cells that respond to infection-causing microbes (pathogens) by initiating and coordinating both local and body-wide immunity. These cells can detect and distinguish between viruses, bacteria, or fungi. Activation of specific macrophages genes against each microbe must be correct for a proper immune response.

“The macrophage is capable of responding to different types of pathogens and mounting different kinds of defenses,” Alexander Hoffmann, PhD, a professor of microbiology at the University of California, Los Angeles (UCLA) and the study’s senior author, said in a press release.

“The defense units — army, navy, air force, special operations — are mediated by groups of genes. For each immune threat, the right groups of genes must be mobilized. That requires precise and reliable communication with those units about the nature of the threat,” Hoffmann said.

NF-kappaB — a protein complex that controls gene activity — plays a crucial role in regulating immune responses in macrophages. Several mutations have been associated with the abnormal regulation of NF-kappaB in people with Sjögren’s, but how these genetic changes affect NF-kappaB function remains unknown.

To learn how macrophages distinguish between different types of microbes, Hoffmann and his team, based at UCLA, used a unique imaging technique to track the movement of NF-kappaB within single macrophages isolated from the bone marrow of healthy mice in response to different immune stimuli.

The immune stimuli included the signaling protein TNF (cytokine) as well as molecules derived from the bacteria S. pneumoniae (Pam3CSK4), M. tuberculosis (CpG), and E. coli (LPS), and also rotavirus A (poly(I:C)). Each pathogen molecule was incubated with 300 to 600 single macrophages at four to seven different concentrations.

This generated more than 12,000 single-cell trajectories, captured by more than three million cell images over 21 hours that traced the movement of NF-kappaB into the cell nucleus over time in response to each stimulus.

Initial analysis revealed over 900 patterns of NF-kappaB movement (activity). Some included a wave-like pattern (oscillation) of NF-kappaB in response to TNF, in which NF-kappaB increased and decreased multiple times within the nucleus over time. In contrast, in response to LPS from E. coli, NF-kappaB initially increased, then gradually decrease over time without oscillating.

Using a computer algorithm, the team searched for features that appeared when macrophages responded to each stimulus. They discovered six specific dynamic features — referred to as “words” or signaling codons — that most frequently correlated with each response.

“The six [NF-kappaB] dynamical features, identified as conveying information about the extracellular stimulus to the nucleus, represent potential codewords of the temporal [NF-kappaB] signaling code and are referred to as [NF-kappaB] signaling codons,” the team wrote. 

The six signaling codons are the speed of NF-kappaB activation, the height of the peak of wave-like NF-kappaB activity, the suppression of NF-kappaB activity, the accumulation of activity at a late time point, the degree of immediate activity, and the duration of NF-kappaB activity above a low threshold.

For example, in response to Pam3CSK4 and LPS-triggered signaling, NF-kappaB movement into the nucleus was rapid but slower in response to CpG and poly(I:C). For Pam3CSK4, CpG, and LPS, the peak of wave-like NF-kappaB activity was high but lower for TNF and poly(I:C). 

Next, a machine-learning algorithm was applied to model the immune response of macrophages by teaching the computer the six signaling codons. The algorithm was able to recognize each stimulus correctly. In contrast, when five signaling codons were used, the computer was less accurate.

The team then tested macrophages isolated from the bone marrow of a Sjögren’s mouse model that mimicked the genetic variants associated with poor NF-kappaB regulation found in human patients. 

Unlike macrophages from healthy mice, these Sjögren’s syndrome macrophages showed a lack of wave-like NF-kappaB patterns in response to all stimuli. In particular, the specificity of the “oscillatory” codon was diminished in these macrophages, as well as the specificity of the “duration” and the “early versus late” codon.

Applying these findings to the machine learning tool showed the ability of Sjögren’s macrophages to identify the proper stimuli was greatly diminished, as seen by an increase in the false positive and false discovery rates for TNF and LPS. Furthermore, the ability to distinguish TNF and poly(I:C) was also reduced compared with the results from healthy mice.

“These analyses indicate that [Sjögren’s] macrophages have diminished ability to generate stimulus-specific [NF-kappaB] signaling dynamics and suggest that signaling codon confusion and mistranslation may play a role in the etiology [cause] of sporadic inflammatory diseases,” the researchers wrote. 

“Indeed, we found defects in the use of two of these words,” Hoffmann said. “It’s as if instead of saying, ‘Respond to attacker down the street,’ the cells are incorrectly saying, ‘Respond to attacker in the house.’”

An analysis of genes potentially affected by these alterations suggested that the confusion of NF-kappaB codons led to the inappropriate expression of type I interferon, an immune regulatory molecule associated with Sjögren’s syndrome. 

“In this work, we report the identification of six dynamical features that characterize complex, stimulus-specific time-course trajectories of [NF-kappaB] activities in single primary macrophage cells,” the scientists wrote. “Macrophages derived from a mouse model of the systemic inflammatory disease, Sjögren’s syndrome, showed increased levels of ligand confusion.”

“Cells have evolved an immune response code, or language,” Hoffmann said. “We have identified some words in that language, and we know these words are important because of what happens when they are misused. Now we need to understand the meaning of the words, and we are making rapid progress.”

“It’s as exciting as when archeologists discovered the Rosetta stone and could begin to read Egyptian hieroglyphs,” he added.