# Systems analysis of cell-to-cell variability and paracrine signaling motifs regulating TLR activation

> **NIH NIH R01** · YALE UNIVERSITY · 2020 · $297,768

## Abstract

Project Summary
A central question in biology is, How do populations of cells execute rapid, reproducible, and coordinated
responses despite variability in gene expression across isogenic cells? One way that cells coordinate
responses to environmental cues is through intermediate extracellular signals that are secreted and sensed by
the cells; but because most studies of secreted extracellular signals are in cell populations, it is not known if
these signals are produced uniformly or vary at a single-cell level. Recent studies of the toll-like receptor
(TLR)-induced inflammatory response–completed by the applicants' lab and others–strongly support a new
regulatory motif by which this response is coordinated by subsets of heterogeneously secreting cells that
dynamically reprogram the population. The objective of this proposal is to dissect the mechanisms of this novel
regulatory motif in TLR-stimulated macrophages. The central hypothesis is that subset(s) of `first responder'
cells that are regulated by differences in cell signaling state coordinate the inflammatory response via
paracrine signaling to neighboring cells. The rationale for the proposed research is that understanding how
paracrine communication between subsets of cells controls the overall inflammatory response will open up new
therapeutic strategies that target the balance between these functional subsets to more specifically treat
chronic inflammatory and autoimmune disorders linked to dysregulated TLR signaling. Guided by the
preliminary data, the central hypothesis will be tested by pursuing three specific aims. In Aim 1, a novel assay
to measure multiplexed secretion in single cells will be used to quantify cell-to-cell heterogeneity in secretion
and to determine which inflammatory functions depend on paracrine communication; data-driven statistical
models built from single-cell data sets will then be used to hypothesize and test regulatory relationships
between extracellular signals. Aim 2 will use live-cell imaging of intracellular signaling dynamics in single cells
to determine if cell state controls the range of observed inflammatory gene expression levels across individual
cells. In Aim 3, a computational model of cell-cell communication fit to single-cell data will be used to explore
the advantage of using diverse cell responses versus more homogeneous ones to coordinate the activation
and resolution of the inflammatory response; model predictions will then be tested experimentally. The
contribution of the proposed research will be to show that cell-cell communication between heterogeneous
responder cells indeed represents a new regulatory motif for coordinating responses to external stimuli. The
approach is innovative because it combines single-cell technologies to experimentally quantify signaling and
secretion in individual cells with computational modeling to interpret these complex data sets. This research
will be significant because it is expected that similar mechan...

## Key facts

- **NIH application ID:** 9878121
- **Project number:** 5R01GM123011-04
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** KATHRYN MILLER-JENSEN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $297,768
- **Award type:** 5
- **Project period:** 2017-03-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9878121

## Citation

> US National Institutes of Health, RePORTER application 9878121, Systems analysis of cell-to-cell variability and paracrine signaling motifs regulating TLR activation (5R01GM123011-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9878121. Licensed CC0.

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