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Open Access Publications from the University of California

Scholarly Works (18 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2021)

Innate immune sentinels serve to both safeguard the organism from foreign invaders and appropriately manage internal damage signals. These cells are distributed throughout the body – circulating the blood, lining the mucosal membranes and the blood vessels, surveying the connective tissue. Their role is to sense the environment, and then decide whether to respond and how best to respond, to maintain the health of the organism. While sentinels include several different cell types, including fibroblasts, endothelial cells, macrophages, and dendritic cells, we here focus on macrophages and examine two properties that may define their sentinel function: response-specificity and epigenomic programmability.

Immune sentinel macrophages initiate the immune response and interact with T-cells, B-cells, NK cells, etc. to coordinate downstream immune system activation. Considering the thousands of potential human pathogens, hundreds of cell damage molecules, and dozens of host-cytokines that may signify inflammatory danger, it should be evolutionarily unsurprising that macrophages respond differently to each signal. However, quantifying this specificity requires single cell data that reveals response distributions. In Chapter 2, we measured single-cell transcriptomic profiles of macrophages responding to diverse bacterial, viral, and cytokine stimuli to quantify the response-specificity of macrophages. Using information theory, we uncovered distinct functional gene groups that differed in their degree of response-specificity. We found that response-specificity was context-dependent, as some genes lost specificity when macrophages were responding from IFNγ or IL4 cytokine contexts. Examining peritoneal macrophages from old and obese mice, which may harbor inflammatory microenvironments, revealed diminished response-specificity compared to healthy mice. These findings suggest that macrophage response-specificity quantified by single cell mRNA measurements after stimulation may be the basis for an innate immune health score.

Beyond responding to an immediate threat by deploying the appropriate transcriptomic programs, immune sentinels may also retain a stimulus-specific memory of the ligand encountered. Prior experimental studies suggested that stimulus-activated transcription factors when bound to DNA could provide the trigger for epigenomic reprogramming, but the mechanisms remained unclear. In Chapter 3, we developed mathematical models of the nucleosome to investigate mechanisms by which the spontaneous kinetics of nucleosomal-DNA interactions interfaced with the stimulus-specific dynamics of transcription factor activity. First focusing on the signal-dependent transcription factor NFκB, our model predicted that presence or absence of oscillations in NFκB signaling was the primary determinant of alterations to epigenomic state. We next developed more detailed Markov models of the nucleosome with base-pair resolution. Fitting the models to time-course stimulus-response ATAC-seq data, we found that kinetic rates of nucleosome unwrapping were slower in vivo than reported in vitro rates, and that the propensity for nucleosome eviction was greatest if transcription factor binding sites sat close to but not on the nucleosome dyad. This work uncovered biophysical principles governing nucleosome dynamics in vivo, enabling the prediction of epigenomic alterations during inflammation at single nucleosome resolution.

    Cover page: Response Specificity and Epigenomic Programmability in Innate Immune Sentinels
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2022)
    Macrophages are first responders for the immune system. In this role, they have both effector functions for neutralizing pathogens and sentinel functions for alerting other immune cells of diverse pathologic threats, thereby initiating and coordinating a multipronged immune response. Macrophages are distributed throughout the body-they circulate in the blood, line the mucosal membranes, reside within organs, and survey the connective tissue. Several reviews have summarized their diverse roles in different physiological scenarios and in the initiation or amplification of different pathologies. In this review, we propose that both the effector and the sentinel functions of healthy macrophages rely on three hallmark properties: response specificity, context dependence, and stimulus memory. When these hallmark properties are diminished, the macrophage's biological functions are impaired, which in turn results in increased risk for immune dysregulation, manifested by immune deficiency or autoimmunity. We review the evidence and the molecular mechanisms supporting these functional hallmarks.
      Cover page: Functional Hallmarks of Healthy Macrophage Responses: Their Regulatory Basis and Disease Relevance
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2019)
      Innate immune sentinel cells must initiate and orchestrate appropriate immune responses for myriad pathogens. These stimulus-specific gene expression responses are mediated by combinatorial and temporal coding within a handful of immune response signaling pathways. We outline the scope of our current understanding and indicate pressing outstanding questions. The innate immune response is a first-line defense against invading pathogens and coordinates the activation and recruitment of specialized immune cells, thereby initiating the adaptive immune response. While the adaptive immune system is capable of highly pathogen-specific immunity through the process of genetic recombination and clonal selection, innate immunity is frequently viewed as a catch-all system that initiates general immune activation. In this review, we are re-examining this view, as we are distinguishing between immune sentinel functions mediated by macrophages and dendritic cells and innate immune effector functions mediated by cells such as neutrophils, NK cells, etc. Given pathogen diversity, including modes of entry, replication cycles, and strategies of immune evasion and spread, all successive waves of the immune response ought to be tailored to the specific immune threat, leading us to postulate that immune sentinel functions by macrophages and dendritic cells ought to be highly stimulus-specific. Here we review the experimental evidence for stimulus-specific responses by immune sentinel cells which initiate and coordinate immune responses, as well as the mechanisms by which this specificity may be achieved.
        Cover page: Stimulus-specificity in the responses of immune sentinel cells
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2021)
        Immune sentinel cells initiate immune responses to pathogens and tissue injury and are capable of producing highly stimulus-specific responses. Insight into the mechanisms underlying such specificity has come from the identification of regulatory factors and biochemical pathways, as well as the definition of signaling circuits that enable combinatorial and temporal coding of information. Here, we review the multi-layered molecular mechanisms that underlie stimulus-specific gene expression in macrophages. We categorize components of inflammatory and anti-pathogenic signaling pathways into five layers of regulatory control and discuss unifying mechanisms determining signaling characteristics at each layer. In this context, we review mechanisms that enable combinatorial and temporal encoding of information, identify recurring regulatory motifs and principles, and present strategies for integrating experimental and computational approaches toward the understanding of signaling specificity in innate immunity.
          Cover page: Stimulus-specific responses in innate immunity: Multilayered regulatory circuits
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          Immune sentinel macrophages initiate responses to pathogens via hundreds of immune response genes. Each immune threat demands a tailored response, suggesting that the capacity for stimulus-specific gene expression is a key functional hallmark of healthy macrophages. To quantify this property, termed "stimulus-response specificity" (SRS), we developed a single-cell experimental workflow and analytical approaches based on information theory and machine learning. We found that the response specificity of macrophages is driven by combinations of specific immune genes that show low cell-to-cell heterogeneity and are targets of separate signaling pathways. The "response specificity profile," a systematic comparison of multiple stimulus-response distributions, was distinctly altered by polarizing cytokines, and it enabled an assessment of the functional state of macrophages. Indeed, the response specificity profile of peritoneal macrophages from old and obese mice showed characteristic differences, suggesting that SRS may be a basis for measuring the functional state of innate immune cells. A record of this paper's transparent peer review process is included in the supplemental information.
            Cover page: Quantifying stimulus-response specificity to probe the functional state of macrophages
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2020)
            Pathogen-derived lipopolysaccharide (LPS) and cytokine tumor necrosis factor (TNF) activate NFκB with distinct duration dynamics, but how immune response genes decode NFκB duration to produce stimulus-specific expression remains unclear. Here, detailed transcriptomic profiling of combinatorial and temporal control mutants identified 81 genes that depend on stimulus-specific NFκB duration for their stimulus-specificity. Combining quantitative experimentation with mathematical modeling, we found that for some genes a long mRNA half-life allowed effective decoding, but for many genes this was insufficient to account for the data; instead, we found that chromatin mechanisms, such as a slow transition rate between inactive and RelA-bound enhancer states, could also decode NFκB dynamics. Chromatin-mediated decoding is favored by genes acting as immune effectors (e.g., tissue remodelers and T cell recruiters) rather than immune regulators (e.g., signaling proteins and monocyte recruiters). Overall, our results delineate two gene regulatory strategies that decode stimulus-specific NFκB dynamics and determine distinct biological functions.
              Cover page: Gene Regulatory Strategies that Decode the Duration of NFκB Dynamics Contribute to LPS- versus TNF-Specific Gene Expression
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2021)
              Macrophages initiate inflammatory responses via the transcription factor NFκB. The temporal pattern of NFκB activity determines which genes are expressed and thus, the type of response that ensues. Here, we examined how information about the stimulus is encoded in the dynamics of NFκB activity. We generated an mVenus-RelA reporter mouse line to enable high-throughput live-cell analysis of primary macrophages responding to host- and pathogen-derived stimuli. An information-theoretic workflow identified six dynamical features-termed signaling codons-that convey stimulus information to the nucleus. In particular, oscillatory trajectories were a hallmark of responses to cytokine but not pathogen-derived stimuli. Single-cell imaging and RNA sequencing of macrophages from a mouse model of Sjögren's syndrome revealed inappropriate responses to stimuli, suggestive of confusion of two NFκB signaling codons. Thus, the dynamics of NFκB signaling classify immune threats through six signaling codons, and signal confusion based on defective codon deployment may underlie the etiology of some inflammatory diseases.
                Cover page: Six distinct NFκB signaling codons convey discrete information to distinguish stimuli and enable appropriate macrophage responses
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2021)
                The epigenome of macrophages can be reprogrammed by extracellular cues, but the extent to which different stimuli achieve this is unclear. Nuclear factor κB (NF-κB) is a transcription factor that is activated by all pathogen-associated stimuli and can reprogram the epigenome by activating latent enhancers. However, we show that NF-κB does so only in response to a subset of stimuli. This stimulus specificity depends on the temporal dynamics of NF-κB activity, in particular whether it is oscillatory or non-oscillatory. Non-oscillatory NF-κB opens chromatin by sustained disruption of nucleosomal histone-DNA interactions, enabling activation of latent enhancers that modulate expression of immune response genes. Thus, temporal dynamics can determine a transcription factor's capacity to reprogram the epigenome in a stimulus-specific manner.
                  Cover page: NF-κB dynamics determine the stimulus specificity of epigenomic reprogramming in macrophages
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2022)
                  The genomic positions of nucleosomes are a defining feature of the cell's epigenomic state, but signal-dependent transcription factors (SDTFs), upon activation, bind to specific genomic locations and modify nucleosome positioning. Here we leverage SDTFs as perturbation probes to learn about nucleosome dynamics in living cells. We develop Markov models of nucleosome dynamics and fit them to time course sequencing data of DNA accessibility. We find that (1) the dynamics of DNA unwrapping are significantly slower in cells than reported from cell-free experiments, (2) only models with cooperativity in wrapping and unwrapping fit the available data, (3) SDTF activity produces the highest eviction probability when its binding site is adjacent to but not on the nucleosome dyad, and (4) oscillatory SDTF activity results in high location variability. Our work uncovers the regulatory rules governing SDTF-induced nucleosome dynamics in live cells, which can predict chromatin accessibility alterations during inflammation at single-nucleosome resolution.
                    Cover page: Stochastic models of nucleosome dynamics reveal regulatory rules of stimulus-induced epigenome remodeling
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2024)
                    Macrophages sense pathogens and orchestrate specific immune responses. Stimulus specificity is thought to be achieved through combinatorial and dynamical coding by signaling pathways. While NFκB dynamics are known to encode stimulus information, dynamical coding in other signaling pathways and their combinatorial coordination remain unclear. Here, we established live-cell microscopy to investigate how NFκB and p38 dynamics interface in stimulated macrophages. Information theory and machine learning revealed that p38 dynamics distinguish cytokine TNF from pathogen-associated molecular patterns and high doses from low, but contributed little to information-rich NFκB dynamics when both pathways are considered. This suggests that immune response genes benefit from decoding immune signaling dynamics or combinatorics, but not both. We found that the heterogeneity of the two pathways is surprisingly uncorrelated. Mathematical modeling revealed potential sources of uncorrelated heterogeneity in the branched pathway network topology and predicted it to drive gene expression variability. Indeed, genes dependent on both p38 and NFκB showed high scRNAseq variability and bimodality. These results identify combinatorial signaling as a mechanism to restrict NFκB-AND-p38-responsive inflammatory cytokine expression to few cells.
                      Cover page: Dynamical and combinatorial coding by MAPK p38 and NFκB in the inflammatory response of macrophages.
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