Follicular lymphoma triggers phenotypic and functional remodeling of the human lymphoid stromal cell landscape.

Lymphoid stromal cells (LSCs) are essential organizers of immune responses. We analyzed tonsillar tissue by combining flow cytometry, in situ imaging, RNA sequencing, and functional assays, defining three distinct human LSC subsets. The integrin CD49a designated perivascular stromal cells exhibiting features of local committed LSC precursors and segregated cytokine and chemokine-producing fibroblastic reticular cells (FRCs) supporting B and T cell survival. The follicular dendritic cell transcriptional profile reflected active responses to B cell and non-B cell stimuli. We therefore examined the effect of B cell stimuli on LSCs in follicular lymphoma (FL). FL B cells interacted primarily with CD49a+ FRCs. Transcriptional analyses revealed LSC reprogramming in situ downstream of the cytokines tumor necrosis factor (TNF) and transforming growth factor ß (TGF-ß), including increased expression of the chemokines CCL19 and CCL21. Our findings define human LSC populations in healthy tissue and reveal bidirectional crosstalk between LSCs and malignant B cells that may present a targetable axis in lymphoma.

Role of cancer-associated fibroblast subpopulations in immune infiltration, as a new means of treatment in cancer.

The tumor microenvironment (TME) has been identified as one of the driving factors of tumor progression and invasion. Within this microenvironment, cancer-associated fibroblasts (CAF) have multiple tumor-promoting functions and play key roles in drug resistance, through multiple mechanisms, including extracellular matrix (ECM) remodeling, production of growth factors, cytokines, and chemokines, and modulation of metabolism and angiogenesis. More recently, a growing body of evidence has shown that CAF also modulate immune cell activity and suppress anti-tumor immune response. In this review, we describe the current knowledge on CAF heterogeneity in terms of identity and functions. Moreover, we analyze how distinct CAF subpopulations differentially interact with immune cells, with a particular focus on T lymphocytes. We address how specific CAF subsets contribute to cancer progression through induction of an immunosuppressive microenvironment. Finally, we highlight potential therapeutic strategies for targeting CAF subpopulations in cancer.

Molecular and Clinical Portrait of HER2-low Invasive Lobular Carcinomas.

Invasive lobular carcinomas (ILCs) have a low frequency of ERBB2 amplification, therefore restricting the use of conventional anti-HER2 therapies for this histologic special type. Conversely, ILCs with low HER2 overexpression may represent a broader target for the use of emerging antibody drug conjugate therapies targeting HER2, since these treatments have proven effective in HER2-low breast cancers. Very scarce data about HER2-low ILCs have been so far published, although these tumors could have different prevalence and histomolecular specificities compared with invasive breast carcinoma of no special type (IBC-NST). Our aims in that context were to decipher the clinicopathological and molecular features of a large series of HER2-low ILCs. Comparative evaluation of HER2-low prevalence was done based on a retrospective series of 7970 patients from Institut Curie, with either primary invasive lobular (N = 1103) or no special type (N = 6867) invasive carcinoma. Clinicopathological and molecular analyses of HER2-zero, HER2-low, and HER2-positive ILCs were performed on a subgroup of 251 patients who underwent surgery for a primary ILC between 2005 and 2008. The mutational profile of these 251 cases was determined from RNAseq data. Compared with HER2-negative IBC-NSTs, the HER2-negative ILCs were found to display a higher frequency of HER2-zero cases (59.4% vs 53.7%) and a lower frequency of HER2-low (40.6% vs 46.3%) (P < .001). Clinicopathological features associated with HER2-low status (vs HER2-zero) in ILC were older age, postmenopausal status, nonclassic ILC histological types, higher grade, proliferation, and estrogen receptor expression levels. Survival curve analysis showed a significantly lower risk of local recurrence for HER2-low (vs HER2-zero) ILCs, but no association was found between HER2 status and either breast cancer-specific survival or distant metastasis-free interval. ERBB3 was the unique mutated gene exclusively associated with HER2-low ILCs yet being mutated at a low frequency (7.1%) (false discovery rate < 0.05). In conclusion, HER2-low ILCs exhibit their own particularities, both on clinical-pathological and molecular levels. Our findings call for larger multicenter validation studies.

Fibroblast heterogeneity in tumor micro-environment: Role in immunosuppression and new therapies.

In tumors, Cancer-Associated Fibroblasts (CAFs) constitute the most prominent component of the tumor microenvironment (TME). CAFs are heterogeneous and composed of different CAF subsets exerting distinct functions in tumors. Specific CAF subpopulations actively influence various aspects of tumor growth, including cancer cell survival and proliferation, angiogenesis, extracellular matrix (ECM) remodeling, metastatic spread and chemoresistance. During the past decade, some CAF subsets have also been shown to modulate anti-tumor immune response. Indeed, they can increase the content in regulatory T lymphocytes and inhibit the activity of effector and cytotoxic immune cells. These functions are mainly controlled by their constitutive secretion of cytokines, chemokines, growth factors and ECM proteins, either directly in the surrounding extracellular space or through micro-vesicles. Some CAFs also express key regulators of immune checkpoints. The different roles played by CAFs, both as immunosuppressor or as physical support for tumor cell progression, set them as promising targets for anti-tumor therapies. In this review, we describe the main current knowledge on CAFs heterogeneity and immunosuppressive microenvironment, as well as their potential therapeutic implications.

Fibroblast heterogeneity in solid tumors: From single cell analysis to whole-body imaging.

Cancer-Associated Fibroblasts (CAFs) represent the most prominent component of the tumor microenvironment (TME). Recent studies demonstrated that CAF are heterogeneous and composed of different subpopulations exerting distinct functions in cancer. CAF populations differentially modulate various aspects of tumor growth, including cancer cell proliferation, extra-cellular matrix remodeling, metastatic dissemination, immunosuppression and resistance to treatment. Among other markers, the Fibroblast Activation Protein (FAP) led to the identification of a specific CAF subpopulation involved in metastatic spread and immunosuppression. Expression of FAP at the surface of CAF is detected in many different cancer types of poor prognosis. Thus, FAP recently appears as an appealing target for therapeutic and molecular imaging applications. In that context, 68Ga-labeled radiopharmaceutical-FAP-inhibitors (FAPI) have been recently developed and validated for quantitatively mapping FAP expression over the whole-body using Positron Emission Tomography (PET/CT). In this review, we describe the main current knowledge on CAF subpopulations and their distinct functions in solid tumors, as well as the promising diagnostic and therapeutic implications of radionuclides targeting FAP.

Definition of a novel breast tumor-specific classifier based on secretome analysis.

BACKGROUND: During cancer development, the normal tissue microenvironment is shaped by tumorigenic events. Inflammatory mediators and immune cells play a key role during this process. However, which molecular features most specifically characterize the malignant tissue remains poorly explored. METHODS: Within our institutional tumor microenvironment global analysis (T-MEGA) program, we set a prospective cohort of 422 untreated breast cancer patients. We established a dedicated pipeline to generate supernatants from tumor and juxta-tumor tissue explants and quantify 55 soluble molecules using Luminex or MSD. Those analytes belonged to five molecular families: chemokines, cytokines, growth factors, metalloproteinases, and adipokines. RESULTS: When looking at tissue specificity, our dataset revealed some breast tumor-specific characteristics, as IL-16, as well as some juxta-tumor-specific secreted molecules, as IL-33. Unsupervised clustering analysis identified groups of molecules that were specific to the breast tumor tissue and displayed a similar secretion behavior. We identified a tumor-specific cluster composed of nine molecules that were secreted fourteen times more in the tumor supernatants than the corresponding juxta-tumor supernatants. This cluster contained, among others, CCL17, CCL22, and CXCL9 and TGF-ß1, 2, and 3. The systematic comparison of tumor and juxta-tumor secretome data allowed us to mathematically formalize a novel breast cancer signature composed of 14 molecules that segregated tumors from juxta-tumors, with a sensitivity of 96.8% and a specificity of 96%. CONCLUSIONS: Our study provides the first breast tumor-specific classifier computed on breast tissue-derived secretome data. Moreover, our T-MEGA cohort dataset is a freely accessible resource to the biomedical community to help advancing scientific knowledge on breast cancer.

Apoptosis mapping in space and time of 3D tumor ecosystems reveals transmissibility of cytotoxic cancer death.

The emerging tumor-on-chip (ToC) approaches allow to address biomedical questions out of reach with classical cell culture techniques: in biomimetic 3D hydrogels they partially reconstitute ex vivo the complexity of the tumor microenvironment and the cellular dynamics involving multiple cell types (cancer cells, immune cells, fibroblasts, etc.). However, a clear bottleneck is the extraction and interpretation of the rich biological information contained, sometime hidden, in the cell co-culture videos. In this work, we develop and apply novel video analysis algorithms to automatically measure the cytotoxic effects on human cancer cells (lung and breast) induced either by doxorubicin chemotherapy drug or by autologous tumor-infiltrating cytotoxic T lymphocytes (CTL). A live fluorescent dye (red) is used to selectively pre-stain the cancer cells before co-cultures and a live fluorescent reporter for caspase activity (green) is used to monitor apoptotic cell death. The here described open-source computational method, named STAMP (spatiotemporal apoptosis mapper), extracts the temporal kinetics and the spatial maps of cancer death, by localizing and tracking cancer cells in the red channel, and by counting the red to green transition signals, over 2-3 days. The robustness and versatility of the method is demonstrated by its application to different cell models and co-culture combinations. Noteworthy, this approach reveals the strong contribution of primary cancer-associated fibroblasts (CAFs) to breast cancer chemo-resistance, proving to be a powerful strategy to investigate intercellular cross-talks and drug resistance mechanisms. Moreover, we defined a new parameter, the 'potential of death induction', which is computed in time and in space to quantify the impact of dying cells on neighbor cells. We found that, contrary to natural death, cancer death induced by chemotherapy or by CTL is transmissible, in that it promotes the death of nearby cancer cells, suggesting the release of diffusible factors which amplify the initial cytotoxic stimulus.

A subset of activated fibroblasts is associated with distant relapse in early luminal breast cancer.

BACKGROUND: Early luminal breast cancer (BC) represents 70% of newly diagnosed BC cases. Among them, small (under 2 cm) BC without lymph node metastasis (classified as T1N0) have been rarely studied, as their prognosis is generally favorable. Nevertheless, up to 5% of luminal T1N0 BC patients relapse with distant metastases that ultimately prove fatal. The aim of our work was to identify the mechanisms involved in metastatic recurrence in these patients. METHODS: Our study addresses the role that autonomous and non-autonomous tumor cell features play with regard to distant recurrence in early luminal BC patients. We created a cohort of T1N0 luminal BC patients (tumors between 0.5-2 cm without lymph node metastasis) with metastatic recurrence ("cases") and corresponding "controls" (without relapse) matched 1:1 on main prognostic factors: age, grade, and proliferation. We deciphered different characteristics of cancer cells and their tumor micro-environment (TME) by deep analyses using immunohistochemistry. We performed in vitro functional assays and highlighted a new mechanism of cooperation between cancer cells and one particular subset of cancer-associated fibroblasts (CAF). RESULTS: We found that specific TME features are indicative of relapse in early luminal BC. Indeed, quantitative histological analyses reveal that "cases" are characterized by significant accumulation of a particular CAF subset (CAF-S1) and decrease in CD4+ T lymphocytes, without any other association with immune cells. In multivariate analysis, TME features, in particular CAF-S1 enrichment, remain significantly associated with recurrence, thereby demonstrating their clinical relevance. Finally, by performing functional analyses, we demonstrated that CAF-S1 pro-metastatic activity is mediated by the CDH11/osteoblast cadherin, consistent with bones being a major site of metastases in luminal BC patients. CONCLUSIONS: This study shows that distant recurrence in T1N0 BC is strongly associated with the presence of CAF-S1 fibroblasts. Moreover, we identify CDH11 as a key player in CAF-S1-mediated pro-metastatic activity. This is independent of tumor cells and represents a new prognostic factor. These results could assist clinicians in identifying luminal BC patients with high risk of relapse. Targeted therapies against CAF-S1 using anti-FAP antibody or CDH11-targeting compounds might help in preventing relapse for such patients with activated stroma.

Obesity-induced activation of JunD promotes myocardial lipid accumulation and metabolic cardiomyopathy.

AIMS: Metabolic cardiomyopathy (MC)-characterized by intra-myocardial triglyceride (TG) accumulation and lipotoxic damage-is an emerging cause of heart failure in obese patients. Yet, its mechanisms remain poorly understood. The Activator Protein 1 (AP-1) member JunD was recently identified as a key modulator of hepatic lipid metabolism in obese mice. The present study investigates the role of JunD in obesity-induced MC. METHODS AND RESULTS: JunD transcriptional activity was increased in hearts from diet-induced obese (DIO) mice and was associated with myocardial TG accumulation and left ventricular (LV) dysfunction. Obese mice lacking JunD were protected against MC. In DIO hearts, JunD directly binds PPARγ promoter thus enabling transcription of genes involved in TG synthesis, uptake, hydrolysis, and storage (i.e. Fas, Cd36, Lpl, Plin5). Cardiac-specific overexpression of JunD in lean mice led to PPARγ activation, cardiac steatosis, and dysfunction, thereby mimicking the MC phenotype. In DIO hearts as well as in neonatal rat ventricular myocytes exposed to palmitic acid, Ago2 immunoprecipitation, and luciferase assays revealed JunD as a direct target of miR-494-3p. Indeed, miR-494-3p was down-regulated in hearts from obese mice, while its overexpression prevented lipotoxic damage by suppressing JunD/PPARγ signalling. JunD and miR-494-3p were also dysregulated in myocardial specimens from obese patients as compared with non-obese controls, and correlated with myocardial TG content, expression of PPARγ-dependent genes, and echocardiographic indices of LV dysfunction. CONCLUSION: miR-494-3p/JunD is a novel molecular axis involved in obesity-related MC. These results pave the way for approaches to prevent or treat LV dysfunction in obese patients.

The role of reactive oxygen species and metabolism on cancer cells and their microenvironment.

Compelling evidence show that reactive oxygen species (ROS) levels are finely regulated in the cell and can act as "second messengers" in response to diverse stimuli. In tumor epithelial cells, ROS accumulate abnormally and induce signaling cascades that mediate the oncogenic phenotype. In addition to their impact on tumor epithelial cells, ROS also affect the surrounding cells that constitute the tumor microenvironment. Indeed, ROS production increases tumor angiogenesis, drives the onset of inflammation and promotes conversion of fibroblast into myofibroblasts. These cells, initially identified upon wound healing, exhibit similar properties to those observed in fibroblasts associated with aggressive adenocarcinomas. Indeed, analyses of tumors with distinct severity revealed the existence of multiple distinct co-existing subtypes of carcinoma-associated fibroblasts (CAFs), with specific marker protein profiling. Chronic oxidative stress deeply modifies the proportion of these different fibroblast subtypes, further supporting tumor growth and metastatic dissemination. At last, ROS have been implicated in the metabolic reprogramming of both cancer cells and CAFs, allowing an adaptation to oxidative stress that ultimately promotes tumorigenesis and chemoresistance. In this review, we discuss the role of ROS in cancer cells and CAFs and their impact on tumor initiation, progression, and metastasis.

Deletion of the activated protein-1 transcription factor JunD induces oxidative stress and accelerates age-related endothelial dysfunction.

BACKGROUND: Reactive oxygen species are major determinants of vascular aging. JunD, a member of the activated protein-1 family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to reactive oxygen species homeostasis in the vasculature remains unknown. METHODS AND RESULTS: Endothelium-dependent vasorelaxation was impaired in young and old JunD(-/-) mice (6 and 22 months old) compared with age-matched wild-type mice. JunD(-/-) mice displayed an age-independent decline in endothelial nitric oxide release and endothelial nitric oxide synthase activity and increased mitochondrial superoxide formation and peroxynitrite levels. Furthermore, vascular expression and activity of the free radical scavengers manganese and extracellular superoxide dismutase and aldehyde dehydrogenase 2 were reduced, whereas the NADPH oxidase subunits p47phox, Nox2, and Nox4 were upregulated. These redox changes were associated with premature vascular aging, as shown by reduced telomerase activity, increased ß-galactosidase-positive cells, upregulation of the senescence markers p16(INK4a) and p53, and mitochondrial disruption. Interestingly, old wild-type mice showed a reduction in JunD expression and transcriptional activity resulting from promoter hypermethylation and binding with tumor suppressor menin, respectively. In contrast, JunD overexpression blunted age-induced endothelial dysfunction. In human endothelial cells, JunD knockdown exerted a similar impairment of the O2(-)/nitric oxide balance that was prevented by concomitant NADPH inhibition. In parallel, JunD expression was reduced in monocytes from old versus young healthy subjects and correlated with mRNA levels of scavenging and oxidant enzymes. CONCLUSIONS: JunD provides protection in aging-induced endothelial dysfunction and may represent a novel target to prevent reactive oxygen species-driven vascular aging.

WNT-dependent interaction between inflammatory fibroblasts and FOLR2+ macrophages promotes fibrosis in chronic kidney disease.

Chronic kidney disease (CKD) is a public health problem driven by myofibroblast accumulation, leading to interstitial fibrosis. Heterogeneity is a recently recognized characteristic in kidney fibroblasts in CKD, but the role of different populations is still unclear. Here, we characterize a proinflammatory fibroblast population (named CXCL-iFibro), which corresponds to an early state of myofibroblast differentiation in CKD. We demonstrate that CXCL-iFibro co-localize with macrophages in the kidney and participate in their attraction, accumulation, and switch into FOLR2+ macrophages from early CKD stages on. In vitro, macrophages promote the switch of CXCL-iFibro into ECM-secreting myofibroblasts through a WNT/ß-catenin-dependent pathway, thereby suggesting a reciprocal crosstalk between these populations of fibroblasts and macrophages. Finally, the detection of CXCL-iFibro at early stages of CKD is predictive of poor patient prognosis, which shows that the CXCL-iFibro population is an early player in CKD progression and demonstrates the clinical relevance of our findings.

Deciphering the spatial landscape and plasticity of immunosuppressive fibroblasts in breast cancer.

Although heterogeneity of FAP+ Cancer-Associated Fibroblasts (CAF) has been described in breast cancer, their plasticity and spatial distribution remain poorly understood. Here, we analyze trajectory inference, deconvolute spatial transcriptomics at single-cell level and perform functional assays to generate a high-resolution integrated map of breast cancer (BC), with a focus on inflammatory and myofibroblastic (iCAF/myCAF) FAP+ CAF clusters. We identify 10 spatially-organized FAP+ CAF-related cellular niches, called EcoCellTypes, which are differentially localized within tumors. Consistent with their spatial organization, cancer cells drive the transition of detoxification-associated iCAF (Detox-iCAF) towards immunosuppressive extracellular matrix (ECM)-producing myCAF (ECM-myCAF) via a DPP4- and YAP-dependent mechanism. In turn, ECM-myCAF polarize TREM2+ macrophages, regulatory NK and T cells to induce immunosuppressive EcoCellTypes, while Detox-iCAF are associated with FOLR2+ macrophages in an immuno-protective EcoCellType. FAP+ CAF subpopulations accumulate differently according to the invasive BC status and predict invasive recurrence of ductal carcinoma in situ (DCIS), which could help in identifying low-risk DCIS patients eligible for therapeutic de-escalation.

Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function.

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.

Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in DSRCTs.

Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive sarcoma driven by the EWSR1::WT1 chimeric transcription factor. Despite this unique oncogenic driver, DSRCT displays a polyphenotypic differentiation of unknown causality. Using single-cell multi-omics on 12 samples from five patients, we find that DSRCT tumor cells cluster into consistent subpopulations with partially overlapping lineage- and metabolism-related transcriptional programs. In vitro modeling shows that high EWSR1::WT1 DNA-binding activity associates with most lineage-related states, in contrast to glycolytic and profibrotic states. Single-cell chromatin accessibility analysis suggests that EWSR1::WT1 binding site variability may drive distinct lineage-related transcriptional programs, supporting some level of cell-intrinsic plasticity. Spatial transcriptomics reveals that glycolytic and profibrotic states specifically localize within hypoxic niches at the periphery of tumor cell islets, suggesting an additional role of tumor cell-extrinsic microenvironmental cues. We finally identify a single-cell transcriptomics-derived epithelial signature associated with improved patient survival, highlighting the clinical relevance of our findings.

Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

Residual ANTXR1+ myofibroblasts after chemotherapy inhibit anti-tumor immunity via YAP1 signaling pathway.

Although cancer-associated fibroblast (CAF) heterogeneity is well-established, the impact of chemotherapy on CAF populations remains poorly understood. Here we address this question in high-grade serous ovarian cancer (HGSOC), in which we previously identified 4 CAF populations. While the global content in stroma increases in HGSOC after chemotherapy, the proportion of FAP+ CAF (also called CAF-S1) decreases. Still, maintenance of high residual CAF-S1 content after chemotherapy is associated with reduced CD8+ T lymphocyte density and poor patient prognosis, emphasizing the importance of CAF-S1 reduction upon treatment. Single cell analysis, spatial transcriptomics and immunohistochemistry reveal that the content in the ECM-producing ANTXR1+ CAF-S1 cluster (ECM-myCAF) is the most affected by chemotherapy. Moreover, functional assays demonstrate that ECM-myCAF isolated from HGSOC reduce CD8+ T-cell cytotoxicity through a Yes Associated Protein 1 (YAP1)-dependent mechanism. Thus, efficient inhibition after treatment of YAP1-signaling pathway in the ECM-myCAF cluster could enhance CD8+ T-cell cytotoxicity. Altogether, these data pave the way for therapy targeting YAP1 in ECM-myCAF in HGSOC.

Oxidative stress contributes to aging by enhancing pancreatic angiogenesis and insulin signaling.

JunD, a transcription factor of the AP-1 family, protects cells against oxidative stress. Here, we show that junD(-/-) mice exhibit features of premature aging and shortened life span. They also display persistent hypoglycemia due to enhanced insulin secretion. Consequently, the insulin/IGF-1 signaling pathways are constitutively stimulated, leading to inactivation of FoxO1, a positive regulator of longevity. Hyperinsulinemia most likely results from enhanced pancreatic islet vascularization owing to chronic oxidative stress. Indeed, accumulation of free radicals in beta cells enhances VEGF-A transcription, which in turn increases pancreatic angiogenesis and insulin secretion. Accordingly, long-term treatment with an antioxidant rescues the phenotype of junD(-/-) mice. Indeed, dietary antioxidant supplementation was protective against pancreatic angiogenesis, hyperinsulinemia, and subsequent activation of insulin signaling cascades in peripheral tissues. Taken together, these data establish a pivotal role for oxidative stress in systemic regulation of insulin and define a key role for the JunD protein in longevity.

Combined ChIP-Seq and transcriptome analysis identifies AP-1/JunD as a primary regulator of oxidative stress and IL-1ß synthesis in macrophages.

BACKGROUND: The oxidative burst is one of the major antimicrobial mechanisms adopted by macrophages. The WKY rat strain is uniquely susceptible to experimentally induced macrophage-dependent crescentic glomerulonephritis (Crgn). We previously identified the AP-1 transcription factor JunD as a determinant of macrophage activation in WKY bone marrow-derived macrophages (BMDMs). JunD is over-expressed in WKY BMDMs and its silencing reduces Fc receptor-mediated oxidative burst in these cells. RESULTS: Here we combined Jund RNA interference with microarray analyses alongside ChIP-sequencing (ChIP-Seq) analyses in WKY BMDMs to investigate JunD-mediated control of macrophage activation in basal and lipopolysaccharide (LPS) stimulated cells. Microarray analysis following Jund silencing showed that Jund activates and represses gene expression with marked differential expression (>3 fold) for genes linked with oxidative stress and IL-1ß expression. These results were complemented by comparing whole genome expression in WKY BMDMs with Jund congenic strain (WKY.LCrgn2) BMDMs which express lower levels of JunD. ChIP-Seq analyses demonstrated that the increased expression of JunD resulted in an increased number of binding events in WKY BMDMs compared to WKY.LCrgn2 BMDMs. Combined ChIP-Seq and microarray analysis revealed a set of primary JunD-targets through which JunD exerts its effect on oxidative stress and IL-1ß synthesis in basal and LPS-stimulated macrophages. CONCLUSIONS: These findings demonstrate how genetically determined levels of a transcription factor affect its binding sites in primary cells and identify JunD as a key regulator of oxidative stress and IL-1ß synthesis in primary macrophages, which may play a role in susceptibility to Crgn.