What happens when macrophages refuse to eat the dead?

Macrophages, a type of immune cells, are an integral part of our body’s defense system. The term macrophage comes from two Greek words – makro meaning big and phagien meaning eat, which makes them the “big eaters”. And boy, do they love to eat! Some things that they like to chomp on include bacteria and other foreign substances, dying and dead cells, and cancer cells, thus, acting as the body’s cleanup system. This process of eating is not only important for defending against foreign pathogens but is also essential for cleaning up cell debris and maintaining normal bodily functions.

Macrophages typically encapsulate their food by surrounding it with cell extensions, then engulf it and digest it. Check out some cool videos of macrophages eating some bacteria here. This process of eating is typically called “phagocytosis”. However, the term for macrophages eating dying cells is called “efferocytosis”. This term is derived from the Latin word efferre which translates to “take to the grave” or “to bury”. When this mechanism of disposal of cellular corpses goes wrong, the rotting dead cells can lead to inflammation that damages the surrounding tissue. This can lead to many diseases, including coronary artery disease, chronic obstructive pulmonary disease, cystic fibrosis, and rheumatoid arthritis. In a recent publication from the Tabas lab, Dr. Kasikara and Columbia postdoc Dr. Schilperoort explore the molecular mechanisms that underlie impaired efferocytosis and how that leads to the formation of dangerous plaques in the arteries that supply blood to your heart. The buildup of these plaques leads to a condition called coronary artery disease which remains the leading cause of deaths in the United States, causing about 1 in 4 deaths.

Significant advances in genomic sequencing in the past few years have led to the discovery of several mutations that are often correlated with the occurrence of coronary artery disease in patients. One of these mutations is in a gene encoding a protein called PHACTR1. However, because the mutation is present in a part of the gene outside of where the protein-coding sequence lies, it was unclear if this mutation disrupted efferocytosis by disrupting the function of PHACTR1. PHACTR1 regulates the ability of various cell types to expand, contract, and move. While the ability of macrophages to execute these motions is required to engulf or eat cells, whether PHACTR1 is involved in this process in macrophages and thereby macrophage efferocytosis was not known. In this study, the authors made two important discoveries. Firstly, they found that PHACTR1 is essential for macrophage efferocytosis. Secondly, they found that the mutation decreases the expression levels of PHACTR1. The authors investigated more and established that PHACTR1 is important for maintaining an activated version of a motor protein called myosin which is required for cellular movement. Thus, lower levels of PHACTR1 hamper the ability of macrophages to eat dead cells by disrupting cellular movement. This contributes to the buildup of dying cells in our arteries and a consequent increase in the risk of heart attack and stroke.

Fig 1. Model depicting the relationship between efferocytosis and risk of coronary artery disease. Reduced levels of efferocytosis lead to insufficient clearance of dead cells and consequent plaque formation in the arteries. Figure adapted from Kasikara, JCI 2021.

The results from this study provide novel insights into the role of PHACTR1, myosin, and other associated proteins in the pathogenesis and progression of coronary artery disease. Before this study was performed, we only knew that there was a correlation between an increased risk of heart disease and a mutation in PHACTR1 gene. The authors performed rigorous experiments and demonstrated that the mutation changes PHACTR1 production and that this causes heart disease. This information is extremely valuable as it provides a basis for designing future therapies. For example, increasing PHACTR1 production artificially may be an effective strategy for treating coronary artery disease. As defective macrophage efferocytosis is also involved in the pathogenesis of many other diseases, this study has direct implications for the discovery of new treatment paradigms for these diseases as well.

Laboratory evolution of a cellular reprogrammer provides a potent path to stem cell generation

The human body has approximately 15 trillion cells, all of which arise from embryonic stem cells which are considered the building blocks of life. Stem cells renew themselves by dividing indefinitely and can also give rise to cells with specialized functions which ultimately end up forming various organs and tissues in our body. This process is called differentiation. Typically, once cells specialize or differentiate, they lose the ability within the body to go back to being stem cells. Given their unique properties, stem cells have become a critical starting point that scientists can tinker with to develop new drugs and therapies. Because of their tremendous value for research, scientists have figured out non-invasive ways to transform differentiated cells into cells with stem cell like properties. These lab-grown cells, called induced pluripotent stem cells or iPSCs, are typically generated by a process called “cellular reprogramming”.

As Dr. Tania Thimraj explains in a recent article, proteins called transcription factors can act as cellular “fixer-uppers” and renovate differentiated cells to look and behave like stem cells. The current state of the art process for making iPSCs involves excess production, also known as overexpression, of the following transcription factors in differentiated cells: Oct4, Sox2, Klf4, and c-Myc (collectively called the “OKSM” cocktail). Despite significant advances in the formulation of this cocktail, there is still a huge margin for improvement in the ability of this cocktail to transform differentiated cells into stem cells. In a recent study performed by Dr. Tan and co-authored by Dr. Malik, the authors propose that the cocktail is not especially effective because the transcription factors were never under any evolutionary selection pressure to produce stem cells. Inspired by this, the authors set out to use evolution in the dish, also known as directed evolution, to make a more efficacious transcription factor cocktail.

Although natural evolution takes place over millions of years, smaller scale evolution can be done in a laboratory setting at much faster timescales. This approach is known as “directed evolution” and has been successfully used by scientists to evolve proteins with new functionalities. This process involves making random mutations in the protein of interest. Then, these mutants undergo a selection process in an appropriate cellular context so that protein variants with desirable properties can be isolated.

In a pioneering study, members of the Jauch lab, including Dr. Malik, used directed evolution to optimize the cellular reprogramming ability of the transcription factor Sox2. Building on this success, the Jauch lab used directed evolution to make ePOU, an enhanced and evolved version of Oct4 which is an integral part of the OKSM cocktail. In the current study for creating ePOU, the authors made random mutations at six functionally important positions in Oct4 and overexpressed the mutant proteins in mammalian cells such that the Oct4 transcription factor activity was tied to the production of a green fluorescent protein representing stem cell transformation.

This innovative study demonstrates that the transformation potential of naturally occurring transcription factors can be drastically enhanced by directed evolution. In addition, this work also provides a framework for future research on transcription factor engineering for cell reprogramming. By providing a faster and more efficient way to produce stem cells, this study has the potential to accelerate various research and therapeutic avenues such as regenerative medicine, drug efficacy and safety testing, and studying human development and disease.

Dr. Vikas Malik is a Postdoctoral Research Fellow in Dr. Jianlong Wang’s lab in the Department of Medicine at Columbia University Medical Center and is a member of CUPS and the Outreach and Communications Committee.

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