If you’ve ever watched a medical drama, chances are that you’ve seen an episode centered around an organ transplant. These transplants are high risk and important – someone may need a new heart, lung, or liver to survive. The surgeries themselves are inherently dangerous – and that is before you account for the risk of allograft (a.k.a. transplant) rejection!

Braza, et al have shown a macrophage activation pathway that leads to allograft rejection and have now tested a novel, myeloid-cell-specific immunotherapy that resulted in long-term transplant acceptance in mice [Braza, M.S. et al Immunity 49(5): 819-828 e816]. This therapy could allow doctors to circumvent the need for long-term damaging immunosuppression and lead to a future with better outcomes for more patients.

Allograft rejection occurs when the immune system of the organ recipient recognizes the transplanted tissue as “non-self” and attacks and destroys it. (This is based on MHC compatibility, see Immunology 101, “What is MHC and why does it matter?) Even with ideal genetic similarity between recipient and donor, recipients are generally placed on life-long regimens of immunosuppressive drugs to prevent their immune systems from mobilizing against their transplanted organs. These come with their own problems, as continuous immunosuppression carries a long list of risks including increased susceptibility to infections and cancer. The drugs themselves can also be toxic. This unfortunate phenomenon of the cure causing damage is often a function of drug transportation.

Many current immunotherapies targeted at transplant rejection focus on deactivating T Cells. Past research has shown T cell activity to be both necessary and sufficient to trigger transplant rejection. Cutting off one or more T Cell activation signals is an effective therapy in the short term, but the accumulated risks of continuous treatment are not ideal. A recent discovery suggests T cells may not be the only culprit.

In fact, Braza et al suggest it is innate immune cells (specifically macrophages) rather than the adaptive immune system that initiate rejection, meaning we’ve missed a key player up to this point.

Macrophages are part of the innate immune system. They are “first responders” that primarily eat up (phagocytose) bodily invaders (See our Immunology 101). Macrophages have other tools, though, and can act in one of two ways. They can either become activated and work to kill intracellular invaders, or they can assume an anti-inflammatory regulatory-type to help modulate overzealous immune responses. These “regulatory macrophages” pose a problem in cancer — tumors can reprogram intratumoral macrophages to regulatory types, muting the body’s immune response against the cancer. However, these regulatory macrophages could prove very useful in preventing allograft rejection.

Researchers investigated the concept of ‘trained immunity’ to determine if macrophages could be trained into regulators. Trained immunity is a mechanism of innate immune memory that is distinct from the usual process of adaptive immune memory.

In this process, certain stimuli effectively prime innate cells of the myeloid lineage to respond more strongly to future stimuli of the same general specificity (Song and Colonna 2017).  In the case of organ transplants, damage to tissues is sterile — meaning there are no invading pathogens for the immune systems to combat. Sterile, or chemically-induced inflammation, is when inflammatory processes occur without biological, pathogenic invasion when tissues are exposed to sterile chemical invaders, such as silica, asbestos, or medically introduced chemicals.

These chemical agents trigger the production of molecules called DAMPS, or self-derived damage-associated molecular patterns. These DAMPS act in much the same way as PAMPS (See The Innate and adaptive Immune System)  and activate innate immune cells to trigger an immune response. In this case, the DAMP activates the immune system against the transplanted organ. Macrophage activation by this pathway can trigger epigenetic changes which boost pro-inflammatory behavior and cytokine production, initiating a process which can lead to allograft rejection.

Knowing this, researchers set out to develop a two-pronged nano-immunotherapeutic approach to thwart pro-inflammatory skewing of allograft-infiltrating macrophages. They targeted two molecules in this approach: mTOR (molecule 1)  and CD40-TRAF6 (molecule 2). Inhibition of molecule 1 would blunt both a) immune cell activation and b) pro-inflammatory cytokine production while inhibition of molecule 2 would block the effect of costimulatory molecules on T-Cells.

Mammalian target of rapamycin, or mTOR, was their first focus. Researchers encapsulated rapamycin (an inhibitor of mTOR) in a lipid shell (creating a drug called mTORi-HDL) and began an in vitro experiment treating human monocytes first with mTORi-HDL and then exposing them to LPS, a bacterially-derived immune activator. Indeed, monocytes that were first treated with mTORi-HDL showed much less inflammatory behavior than those without treatment. Researchers tested these treated monocytes for evidence of epigenetic reprogramming and found that drug treatment had prevented epigenetic changes at the promoter level of four inflammatory genes associated with trained immunity (TNF𝛼, IL-6, HK, PFKP).

Next, researchers fluorescently dyed molecules of mTORi-HDL to determine their natural distribution within the body and with which immune cells they specifically associate. They observed drug accumulation in mouse kidneys, livers, and spleens as well as in the bone marrow. Notably, the drug that had accumulated in these locations had preferentially formed associations with myeloid, or innate, immune cells, rather than T or B cells. They then repeated this experiment in an allograft heart transplant mouse model. High accumulation of mTORi-HDL was found in allograft hearts. These experiments showed promising results — the drug was shown to preferentially accumulate in key areas and to associate with the innate, rather than the adaptive, immune system. So, it targeted well, but could it inhibit inflammatory rejection?

The next step was to determine exactly which myeloid cells the drug was interacting with by flow cytometry. Researchers again observed a strong preference for myeloid cells, but they were also able to determine that mTORi-HDL had a much higher uptake by macrophages than by other myeloid cells.

Then they assessed the outcomes of mTORi-HDL treatment on macrophages. Treatment was shown to increase the accumulation of regulatory macrophages in allograft tissue and to prevent previously observed epigenetic changes from occurring in graft-infiltrating macrophages. To confirm that the effects being shown were truly caused by the drug’s effect on regulatory macrophages, researchers depleted endemic regulatory macrophages on the day of treatment, resulting in early allograft rejection even upon the administration of the mTORi-HDL therapy. However, researchers were able to rescue allograft survival by reintroducing wild type monocytes.

So: Braza et al have a therapeutic that requires macrophages, is preferentially taken in by macrophages, and trains them to a regulatory and transplant-preserving type all by targeting mTOR/molecule 1.

That leaves molecule 2, CD40-TRAF6.  Prior work by Drs. Shen and Goldstein [J. Am. Soc. Nephrol., 2009] revealed that activated macrophages support T-cell mediated graft-reactive immunity. They secrete cytokines and the absence of these cytokines synergizes blocking costimulation (CD40-CD40L) and aids in allograft acceptance. So, if mTORi-HDL turns off the cytokines — can a different molecule block costimulation?

Braza et al encapsulated a CD40-blocking molecule, TRAF6, in a lipid shell, creating TRAF6i-HDL to use in combination with their previously designed mTORi-HDL. Indeed, they found that the combination treatment was far more effective than the mTORi-HDL treatment alone. The combination treatment achieved a greater than 70% allograft survival rate after 100 days in treated mice. This is especially notable because only three doses of the drug were given — one on the day of the transplant, one on the second day after the transplant, and one on the fifth day after the transplant. These doses were sufficient to maintain allograft acceptance 100 days after surgery.

These experiments demonstrate that short-term therapy is capable of producing long-term results without continuous immunosuppression of the patient. Consequently, the two-part approach of targeting both the metabolic and the epigenetic pathways that factor into trained immunity (specifically in macrophages) may have future applications against other autoimmune disorders, chronic inflammation, cardiovascular diseases, and even allergies. Circumventing the requirement for long-term immunosuppression and its side effects in transplants and other autoimmune disorders could be a gateway to a brighter future in medicine.

Reference: Braza, M. S., M. M. T. van Leent, M. Lameijer, B. L. Sanchez-Gaytan, R. J. W. Arts, C. Perez-Medina, P.

Featured Image: “Medical UI” by Dennis Sch\xe4fer is licensed under CC BY-NC 4.0