Investigating the epigenetic basis of monocyte exhaustion memory following sepsis

Sepsis is a leading cause of death worldwide, with most patient mortality stemming from lingering immune
dysfunction in sepsis survivors. A key feature of sepsis-associated immune dysregulation is monocyte
exhaustion, a phenotype of paradoxical pro-inflammatory and immunosuppressive gene expression, impaired
differentiation, and reduced antigen presentation. Monocyte exhaustion can persist for years after sepsis onset,
a result of long-term immune memory. However, the mechanisms controlling such long-term memory remain to
be elucidated. Whereas previous research has conceptualized innate immune memory through diametrically
opposed mechanisms that either promote (train) or restrict (tolerize) monocyte responses, my preliminary data
suggests that exhaustion represents a distinct memory state characterized by unique immune, transcriptional,
and epigenetic features. Therefore, in contrast to the two-state model for innate memory, I hypothesize that
innate memory represents a continuum of states driven by distinct epigenetic patterning, with prolonged, high-
intensity immune stimulation leading to monocyte exhaustion in septic individuals. In Aim 1 of my proposed study,
I will profile the unique transcriptional and epigenetic features defining monocyte exhaustion, as well as employ
integrative modeling to determine how immune stressor strength, duration, and timing influence the
establishment of distinct innate memory states. In Aim 2, given preliminary data showing genome-wide DNA
hypermethylation in exhausted monocytes, I will test the hypothesis that inhibition of DNA demethylation enzyme
TET2 is upstream of these epigenetic changes, and that treatment with TET agonists is a tractable therapeutic
strategy to restore healthy epigenetic memory. Finally, in Aim 3, based on my recent identification of a novel
DNMT3L isoform expressed in septic monocytes, I will test the altered chromatin affinity and regulatory activity
of this isoform and establish its contribution to DNA methylation reprogramming during monocyte exhaustion.
Completion of these proposed Aims will allow me to develop skills in new experimental techniques, including
single-cell RNA sequencing, reduced representation bisulfite sequencing, in vivo mouse sepsis modeling, and
cytometric arrays. Aims 1 and 3 will be pursued during the K99 mentored research phase at Virginia Tech in the
laboratory of Dr. Liwu Li, an expert in the fields of monocyte biology and innate immune memory. Whereas my
previous graduate studies focused on epigenetics and mammalian development, Dr. Li will provide valuable
instruction as I expand into the topics of immunology and hematology. I will also pursue coursework at Virginia
Tech in computational modeling of biological systems while engaging with professional development workshops
covering such topics as scientific communication, mentorship, and R-series proposal development. The goal of
this project is ultimately to pursue a career as an independent biomedical investigator in academic research;
these studies will serve as a foundation for my own research program aimed at identifying the major molecular
players responsible for establishing and maintaining innate immune memory.

Grant Number: 1K99AI197383-01
NIH Institute/Center: NIH
Principal Investigator: Blake Caldwell