Gene Expression In Specific Populations Research Articles

Activation of Distinct Inflammatory Pathways in LR-MDS Is Determined By Genetics

Background Dysregulated innate immune signaling has been identified as a driver of the complex pathophysiology of myelodysplastic syndromes (MDS), predominantly in low risk (LR)-MDS (Basiorka et al., Blood 2016). The pharmacological targeting of inflammation may therefore help to retard disease development. However, since MDS is a heterogeneous disease, a personalized approach is likely to be necessary, requiring a more detailed knowledge of distinct inflammatory patterns and their variation between genetically defined subgroups of LR-MDS patients (Winter et al., J Clin Oncol 2020). Aims The aim of this work was to describe inflammation states in the bone marrow of LR-MDS patients (IPSS-R ≤ 3.5) compared to controls with and without clonal hematopoiesis of indeterminate potential (CHIP) and to high-risk (HR)-MDS patients (IPSS-R ≥ 4). Methods Cryopreserved bone marrow mononuclear cells (BM-MNCs) and corresponding bone marrow plasma samples from non-CHIP controls (n = 15), CHIP individuals (n = 12) and patients with LR-MDS (n = 47) or HR-MDS (n = 14) were obtained from the MDS registry and the BoHemE study (NCT02867085) at the University Hospitals in Dresden and Leipzig. Targeted expression analysis of 11 inflammasome-related genes (S100A9, NLRP3, PYCARD, CASP1, IL1B, IL18, NLRP1, NLRC4, AIM2, CASP3, CASP4) and multiplex cytokine measurements were performed. To investigate heterogeneity in the LR-MDS cohort, a principle component analysis (PCA) was performed and a cumulative gene expression score was calculated as summed log2 fold changes (mean non-CHIP = 0) of all 11 mRNA expression values for each sample. Furthermore, BM-MNCs of selected non-CHIP (n = 3) and CHIP (n = 3) individuals as well as LR-MDS (n = 14) and HR-MDS (n = 5) patients were FACS-sorted for hematopoietic stem and progenitor cells (HSPCs), monocytes, monocytic and polymorphonuclear myeloid-derived suppressor cells (MDSCs) and lymphocytes. The expression of IL1B, IL18, S100A9, NLRP3, PYCARD, CASP1 and NLRC4 was assessed in each population by qRT-PCR. Finally, colony forming assays were used to assess the effect of the IL-1β-neutralizing antibody canakinumab (100 µg/ml) on the hematopoietic potential of healthy CD34+ HSPCs pre-cultured with CD14+ monocytes from patients with SF3B1-mutated (n = 3) or del(5q) (n = 3) LR-MDS. Results The expression of inflammasome-related genes in the bone marrow increased from non-CHIP through CHIP to LR-MDS individuals, with overall levels of inflammasome-related gene expression being 40% higher in LR-MDS than in non-CHIP controls (p = 0.002). The precise signature of inflammation gene expression varied widely within LR-MDS patients. A PCA identified two separable inflammation states with low (cluster 1, n = 24) and high (cluster 2, n = 23) inflammation patterns (Figure 1), with CASP1, PYCARD and NLRC4 contributing most to the PCA dimensions. The clusters were associated with distinct clinical features and disease genotypes: more than 80% of the SF3B1 mutations were found in cluster 1, while all del(5q) cases were in cluster 2 (Figure 1). Compared to del(5q) samples, those with SF3B1-mutation showed lower expression of IL-1β at both mRNA (3.9-fold decrease, p = 0.0005) and protein level (8.3-fold decrease, p = 0.03). Targeted gene expression analysis of sorted cell populations revealed distinct contributions to the differential patterns of inflammation. IL1B, NLRP3, NLRC4 and S100A9 were expressed in all disease states predominantly by monocytes and monocytic MDSCs, consistent with a dominant role for monocytic cells in determining the inflammatory bone marrow environment. However, IL18 mRNA levels were highest in HSPCs. Compared to untreated controls, canakinumab augmented the colony forming activity of healthy HSPCs pre-cultured with monocytes from SF3B1-mutated MDS (+70%, p = 0.002). The effect in co-cultures with del(5q) MDS-derived monocytes was weaker (+56%, p = 0.188), showing that canakinumab efficacy in this assay does not depend on IL-1β expression level alone. Conclusion We conclude that inflammation patterns in LR-MDS are heterogeneous, highlighting the need for personalization of targeted anti-inflammatory therapies currently entering clinical trials. Our analysis suggests that high resolution studies of gene expression in specific populations or single cells will further resolve a spectrum of inflammation states relevant to prognosis and personalized therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P747: LR-MDS IS CHARACTERIZED BY DIFFERENTIAL EXPRESSION OF INFLAMMASOME-RELATED GENES IN SPECIFIC CELL POPULATIONS

Background: Myelodysplastic Syndromes (MDS) are a group of hematopoietic neoplasms characterized by clonal expansion of hematopoietic stem and progenitor cells (HSPC) and peripheral blood cytopenia. MDS can develop from clonal hematopoiesis of indeterminate potential (CHIP) and progress through low-risk (LR) to high-risk (HR)-MDS and further to acute myeloid leukemia (AML). Recent findings emphasize the role of sterile inflammation in the bone marrow (BM) as a driver of neoplastic progression. Our previous analysis of whole BM mononuclear cells (BM-MNC) has revealed distinct expression patterns of inflammasome-related genes, including IL1B and IL18, to be associated with LR-MDS subgroups and clinical features. This raises the prospect of interrupting disease evolution at an early stage by targeted, personalized anti-inflammatory therapy. The development and stratification of such therapies will ultimately require more detailed knowledge of the variety and consequences of inflammation networks in MDS bone marrow. Aims: Our objective was to assess inflammasome-related gene expression levels in individual cell types in the BM of CHIP, LR-MDS and HR-MDS individuals as well as non-CHIP controls in order to determine a) which cell types contribute to the expression of inflammasome-related genes and b) whether specific disease states are associated with consistent patterns of cell-specific gene expression. Methods: Cryopreserved BM-MNC from 3 non-CHIP controls, 3 CHIP individuals, 14 LR-MDS and 5 HR-MDS patients were obtained from the MDS registry and the BoHemE Study (NCT02867085) at the University Hospitals in Dresden and Leipzig. Hematopoietic stem and progenitor cells (HSPC), monocytes, monocytic myeloid-derived suppressor cells (M-MDSC), polymorphonuclear MDSC (PMN-MDSC), B lymphocytes, T lymphocytes and CD45- cells (control) were sorted from thawed BM-MNC on a BD FACS Jazz. RNA was isolated and the gene expression of IL1B, IL18, S100A9, IRAK4, NLRP3, PYCARD, CASP1 and NLRC4 was assessed by RT-qPCR. Results: The majority of the inflammasome-related genes, including IL1B, NLRP3, NLRC4 and S100A9 were expressed in all disease states predominantly by monocytes and M-MDSC (p<0.001). However, IL18 was expressed at the highest level in HSPC. Consistent to our previous analysis of whole BM-MNC, we found an association between cell-specific gene expression and LR-MDS genetics, with HSPC-derived IL18 mRNA being highest in SF3B1-mutated LR-MDS, while monocytic IL1B mRNA was highest in del(5q) LR-MDS. Furthermore, an apparent progression in expression from non-CHIP through to LR-MDS was detected, with mRNA levels of IL1B and NLRP3 in monocytic cells and PYCARD and CASP1 in HSPC increasing from non-CHIP to CHIP between 1.5- and 7.9-fold. From CHIP to LR-MDS, mRNA levels of CASP1 and NLRC4 in monocytic cells and PYCARD both in HPSC and in monocytic cells increased by 1.8- to 2.1-fold. Summary/Conclusion: We identify features of inflammasome-related gene expression in individual cell populations and disease states reflecting the progression from non-CHIP through CHIP to MDS. Our analysis suggests strongly that high resolution studies of gene expression in specific populations or single cells will resolve a spectrum of MDS-related inflammation states relevant to prognosis and personalized therapy. Therefore, we are currently performing RNA-Seq from sorted cell populations in order to extend our analysis beyond the core inflammasome reported here.

A calcium- and light-gated switch to induce gene expression in activated neurons.

Despite recent advances in optogenetics, it remains challenging to manipulate gene expression in specific populations of neurons. We present a dual-protein switch system, Cal-Light, that translates neuronal-activity-mediated calcium signaling into gene expression in a light-dependent manner. In cultured neurons and brain slices, we show that Cal-Light drives expression of the reporter EGFP with high spatiotemporal resolution only in the presence of both blue light and calcium. Delivery of the Cal-Light components to the motor cortex of mice by viral vectors labels a subset of excitatory and inhibitory neurons related to learned lever-pressing behavior. By using Cal-Light to drive expression of the inhibitory receptor halorhodopsin (eNpHR), which responds to yellow light, we temporarily inhibit the lever-pressing behavior, confirming that the labeled neurons mediate the behavior. Thus, Cal-Light enables dissection of neural circuits underlying complex mammalian behaviors with high spatiotemporal precision.

Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish.

A number of genes affecting axonal projections are currently being identified in zebrafish mutant screens. Analyzing the expression of these genes in the adult brain in relation to specific neuronal populations could yield insights into new functional contexts, such as the successful axonal regeneration in adult zebrafish. Here, we provide a relatively simple procedure for non-radioactive in situ hybridization in sections of adult zebrafish brains in combination with retrograde axonal tracing using the fluorescent neuronal tracer rhodamine dextran amine (RDA). A lesion is inflicted on the spinal cord of adult zebrafish and a crystal of RDA is then applied to the lesion site resulting in retrograde labeling of neurons in the brain through their spinal axons. Six to eighteen days later fish are perfusion-fixed, and in situ hybridization is carried out on vibratome-cut floating sections using a protocol simplified from that used for whole-mounted zebrafish embryos. This procedure leads to robust double labeling of axotomized neurons with RDA and an in situ hybridization signal for the growth-associated protein 43 (GAP-43). This method can be used to identify gene expression in specific populations of projection neurons and to detect changes in gene expression in axotomized neurons in the CNS of adult zebrafish.

Expression of the ceruloplasmin gene in the human retina and brain: implications for a pathogenic model in aceruloplasminemia.

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism characterized by progressive neurodegeneration of the retina and basal ganglia in association with inherited mutations of the ceruloplasmin gene. To begin to elucidate the pathogenesis of this disease, ceruloplasmin gene expression was examined in human brain and retinal tissue. RNA blot analysis and RNAse protection studies demonstrate ceruloplasmin-specific transcripts in multiple regions of the human brain, and biosynthetic studies reveal ceruloplasmin synthesis and secretion in these same regions. Consistent with these observations, in situ hybridization of central nervous system tissue utilizing ceruloplasmin cRNA probes reveals abundant ceruloplasmin gene expression in specific populations of glial cells associated with the brain microvasculature, surrounding dopaminergic melanized neurons in the substantia nigra and within the inner nuclear layer of the retina. Taken in the context of the clinical and pathological features observed in patients with aceruloplasminemia, these data reveal that glial cell-specific ceruloplasmin gene expression is essential for iron homeostasis and neuronal survival in the human central nervous system.

Fos: An immediate‐early transcription factor in neurons

In the past several years a great deal of evidence has accumulated linking neuronal activation events to the regulation of gene expression. We have pursued an analysis of c-fos regulation in the nervous system to elucidate the molecular mechanisms involved in stimulus-transcription coupling. The c-fos gene can be viewed as an archetype of the set of cellular immediate-early genes encoding transcription factors. These genes are believed to function in coupling short-term signals elicited by extracellular to long-term changes in cellular phenotype by orchestrating alterations in target gene expression. Several animal seizure models have been used to demonstrate the activation of gene expression in specific populations of neurons. Using a transgenic mouse approach, based on a foslacZ fusion gene, we now demonstrate an association between c-fos expression and cell death in the nervous system. A delayed and protracted induction was observed following surgical lesion and in response to neurotoxin exposure. This system allows us to determine, for the first time, the DNA regulatory sequences that are responsible for the induction of gene expression in neurons in vivo. Furthermore, foslacZ transgenic mice provide a unique resource for identifying cell populations that respond to specific stimuli or that are susceptible to particular toxins.