Macrophages are white blood cells that play a key role in helping to protect us against microscopic threats, from infections to cancer. These cells constantly survey the body’s tissues and consume pathogens, debris, dead cells and cancer. To do this they scan for “eat me” signals such as IgG antibodies.
It’s critical that patrolling macrophages ignore healthy cells, otherwise they could trigger an autoimmune response. In an in vitro study designed to help understand how macrophages choose what and when to eat, researchers at UC Santa Barbara (UCSB) programmed these immune cells to respond to light, offering up the opportunity to investigate how encounters with cancer cells changed the macrophages’ appetite. They found that they could “prime” the macrophages to subsequently eat more IgG-bound human cancer cells.
“We discovered that giving macrophages an appetizer makes them hungrier for their next meal,” said Meghan Morrissey, PhD, an assistant professor in the department of molecular, cellular, and developmental biology. The team suggests their results could point to a new way to increase the effectiveness of cancer immunotherapies that harness macrophages to combat the disease. The findings also present a more complex account of trained immunity, a kind of memory exhibited in the innate immune system that scientists have only recently recognized.
Morrissey is senior author of the researchers’ published paper in , titled “ .” In their report the team concluded, “Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.” While monitoring the body, macrophages scout for cells and debris tagged with the antibody IgG by other immune cells.
These function as “eat me” signals to the macrophages, which detect them via Fc receptors (FcR) embedded in their cell membrane. Asking “What affects macrophage appetite?” the authors commented, “One important parameter is how sensitive a macrophage is to ‘‘eat-me’’ signals.” Fc receptors are mobile and they begin to cluster when activated by IgG.
This needs to reach a certain threshold for the macrophage to then engulf the target. “Antibody-dependent phagocytosis requires the coordinated activation of a sufficient number of FcRs,” the team explained. “Targets with a subthreshold amount of IgG are not phagocytosed, despite triggering the initial steps in the phagocytosis signaling pathway.
” To enable more detailed investigation of how prior IgG exposure might affect macrophage appetite, lead author Annalise Bond, a doctoral student in Morrissey’s lab, developed an optogenetic approach to cluster the FcR that didn’t require IgG. “To precisely control the temporal pattern of FcR activation across an entire field of cells, we sought to design an optoFcR that could be turned on and off with light,” the investigators explained. With help from UCSB professor Max Willson, PhD, Bond designed a synthetic protein containing part of the FcR receptor fused to cryptochrome 2 (CRY2).
This protein clusters together when activated by blue light, enabling Bond to precisely control the system and trigger the FcR at will. Bond was able to use light to coax the macrophages to consume silica beads coated with a lipid membrane to mimic cancer cells. All without any IgG.
Now the investigators could give the macrophages a “light snack” to see how it affected their eating habits later on. The investigators stimulated engineered macrophages with light, then made the cells wait for different periods of time. They then presented the cells with the mock cancer cells, this time displaying that IgG “eat me” antibody.
They found that the light-activated cells ate much more after their simulated snack when compared with control cells that lacked light-activated FcR. “I’ve described it as Hungry Hungry Hippos,” Bond said, “because they’re just gobbling up everything that’s there.” Activating FcR with subthreshold levels of the IG antibody on cancer cells also primed the macrophages for their next meal.
However, with too much stimulation, the effect disappeared. “If the macrophages got so much IgG that they actually eat, then it wasn’t an appetizer,” Morrissey explained. “It was more like a meal.
So they weren’t hungry anymore.” The authors aren’t positive why macrophages behave this way, but hypothesise that as a macrophage scouts around healthy tissue its top priority is to avoid triggering autoimmunity. So the macrophage sets a pretty high activation threshold.
Now consider a macrophage that begins to encounter IgG antibodies. “Once you see a hint that something’s wrong, now your top priority is clearing the infection, and you’d be willing to damage the tissue a little bit if you had to,” Morrissey said. The macrophages’ appetite peaked around an hour after the initial trigger, before dipping and then rising again for a sustained period after four hours.
“We decided to carefully assay when macrophage priming occurred,” the scientists noted. “To do this, we activated the optoFcR and varied the time before presenting a second stimulus of IgG-coated beads and measuring phagocytosis. We saw robust priming occurring in two discrete waves: a short-term response that peaks around 1 h after FcR activation and a long-term response that begins at 4 h after FcR activation and persists for at least 72 h.
” Bond was curious to try and understand the mechanisms that underlie this pattern. “One hour is way too fast for the cell to make new proteins,” Morrissey said, so something else must be going on. Experiments showed that the macrophages retained their short-term priming when the investigators blocked protein synthesis, suggesting that something else controlled this response.
“Blocking new protein synthesis did not significantly reduce priming at 1 h post stimulation, suggesting that short-term memory is not reliant on new protein production,” the authors stated. However, disabling protein synthesis eliminated the cells’ long-term enhanced appetite, indicating that this behavior relies on changes to gene expression and protein synthesis. “Overall this suggests that there are two mechanisms for macrophage priming—one that operates on a short timescale and does not require synthesis of new proteins and one that operates on a long timescale and requires changes in gene expression,” the scientists wrote.
Additional studies indicated that sub-threshold activation of FcR triggered changes to how the receptors move around the cell membrane. It increases the receptors’ mobility, enabling them to aggregate more easily when exposed to IgG within about an hour. At the same time, the cell begins upregulating different genes and producing new proteins, explaining the longer term effects.
The results of these studies, the team reported “...
suggest that there is an increase in the mobile fraction of FcRs on primed macrophages, which may increase formation of FcR signaling clusters and the probability of initiating phagocytosis.” Morrissey added, “This short-term mechanism is really interesting because it’s a totally different type of immune memory than what’s been seen before.” Macrophages find antibodies like IgG irresistible; they’ll eat pretty much anything tagged with them, even the glass beads Bond used in her experiments.
As a result, monoclonal antibodies have become a popular treatment for various diseases. In fact, antibodies are currently used in many different cancer therapies. Bond was able to increase the efficacy of the antibody Rituximab that is used to treat lymphoma.
Bond and Morrissey’s results suggest that multiple, small doses of antibody therapy will be more effective than a single large dose, since previous doses can prime the cells for the next treatment. Indeed, oncologists have found this to be true through trial and error. “Interestingly, more frequent low-dose treatments of rituximab are more effective at treating chronic lymphocytic leukemia (CLL) patients than higher-dose treatments,” the team pointed out.
There are also other macrophage therapies that might benefit from pretreatment: exposing engineered macrophages used in certain therapies to IgG before introducing them to the patient so they are primed to consume more cancer cells. Asking how it might be possible to “engineer hungrier macrophages to attack cancer cells?” the authors stated, “Our studies reveal that macrophage priming could enhance phagocytosis or other anti-cancer signaling pathways in these macrophages.” For a long time, biologists and doctors thought only the adaptive branch of the immune system had any sort of immunological memory.
But a more nuanced picture has begun to emerge. The newly reported work showed that even parts of the immune system that aren’t commonly thought of as having memory might respond to prompts. And it suggests that immunological memory is a spectrum, with some cells reacting to the here and now; others remembering infections for decades; and some, like macrophages, falling in between.
The work also presents a more complex portrayal of macrophages, suggesting that they’re more sophisticated decision makers than scientists had thought. “In vivo, there are many specialized macrophage sub-populations,” the scientists stated. “Future studies will need to determine whether IgG primes all of these populations using a similar mechanism or whether there are tissue-specific differences.
Future studies should also determine whether therapeutic monoclonal antibodies prime macrophages in vivo or in human tissues.” Morrissey also noted, “Macrophages need to think about the situation they’re in. Are they in healthy tissue and need to avoid autoimmunity, or are they fighting an infection and need to go out guns blazing?” Macrophages actually have two versions of the Fc receptor: One promotes their appetite while the other inhibits it.
And both are triggered by IgG. Macrophages have more of the activating version, so that one eventually wins out. But it’s not clear why the cell has both, rather than just a smaller number of activating FcR.
It’s a mystery Bond is also working to solve. The technique she developed enables her to selectively trigger just one FcR, so in future work she might just be able isolate the role the inhibitory FcR plays. The authors commented, “The FcR family includes activating and inhibiting receptors.
The optoFcR models activating receptors, so future studies will need to address the role of inhibitory FcRs in IgG priming.” Bond added, “Now that I have this tool kit to explore macrophage appetite, I am really interested in understanding how the inhibitory FcR functions..
