Single-cell Transcriptomic Analysis Reveals Rich Pituitary–immune Interactions Under Systemic Inflammation
Ting Yan, Ruiyu Wang, Jingfei Yao, Minmin Luo
Abstract
The pituitary represents an essential hub in the hypothalamus–pituitary–adrenal (HPA) axis. Pituitary hormone-producing cells (HPCs) release several hormones to regulate fundamental bodily functions under normal and stressful conditions. It is well established that the pituitary endocrine gland modulates the immune system by releasing adrenocorticotropic hormone (ACTH) in response to neuronal activation in the hypothalamus. However, it remains unclear how systemic inflammation regulates the transcriptomic profiles of pituitary HPCs. Here, we performed single-cell RNA-sequencing (scRNA-seq) of the mouse pituitary and revealed that upon inflammation, all major pituitary HPCs respond robustly in a cell type-specific manner, with corticotropes displaying the strongest reaction. Systemic inflammation also led to the production and release of noncanonical bioactive molecules, including Nptx2 by corticotropes, to modulate immune homeostasis. Meanwhile, HPCs up-regulated the gene expression of chemokines that facilitated the communication between the HPCs and immune cells. Together, our study reveals extensive interactions between the pituitary and immune system, suggesting multifaceted roles of the pituitary in mediating the effects of inflammation on many aspects of body physiology.
Introduction
As the master gland of the endocrine system and a key component in the hypothalamus–pituitary–adrenal (HPA) axis, the pituitary releases several hormones to regulate numerous physiological functions, such as development, sexual maturation, reproduction, gestation, metabolism, lactation, and stress handling [1]. It also participates in controlling immune responses to inflammatory stimuli [2]. During systemic inflammation that is caused by bacterial or viral infections such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [3–5], the HPA axis orchestrates the release of glucocorticoids to restrain inflammation [6]. The current model suggests that, upon peripheral immune stimulation, activated immune cells produce proinflammatory cytokines, which stimulate vagus nerve activity and in turn activate the paraventricular nucleus (PVN) in the hypothalamus [7,8]. Activation of PVN neurons leads to the release of corticotropin-releasing hormone (CRH) into the anterior pituitary, where corticotropes are triggered to release ACTH [9]. Through general circulation, ACTH travels to the adrenal glands to initiate steroidogenesis and the release of glucocorticoids, which finally prevent the immune system from overreaction [10,11].
Materials and methods
Mice
Animal care and use were approved by the Animal Care and Use Committee of the National Institute of Biological Sciences, Beijing, China (Approval ID: NIBS2022M0036), in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals of China. Male C57BL/6 wild-type mice (8 to 12 weeks old) were obtained from the Beijing Vital River Laboratory Animal Technology. Mice were on a 12-h on, 12-h off-light cycle (lights off at 8 AM and on at 8 PM).
Cell lines
HEK 293T cells (CRL-3216, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (0099–141, Gibco) and 1% penicillin-streptomycin (P/S) (15140–122, Gibco). AtT-20 cells (CCL-89, ATCC) were cultured in F-12K medium supplemented with 15% horse serum (HS) (26050088, Thermo Fisher Scientific), 2.5% FBS, and 1% P/S. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.
Results
Transcriptomic characterization of the pituitary under systemic inflammation
To explore the response of the pituitary cells to immune challenges, we first performed bulk RNA sequencing on pituitary tissues extracted from mice that were intraperitoneally (i.p.) injected with a medium-dose lipopolysaccharide (LPS; 5 mg/kg) or underwent mid-grade cecal ligation and puncture (CLP) (Fig 1A and S1 Table), both of which are commonly used to induce systemic inflammation related to bacterial infections [17]. Up-regulated and down-regulated genes exhibited a high degree of similarity between LPS and CLP treatment (S1A Fig). Principal component analysis (PCA) of the transcriptomic profiles revealed that both LPS and CLP treatments led to clusters distinct from sham and saline controls, with the LPS treatment producing more consistent responses (Figs 1B and S1B). Gene Set Enrichment Analysis (GSEA) revealed strong up-regulation in the expression of genes involved in the signaling pathways related to apoptosis, proinflammatory response, cytokine production, and myeloid leukocyte migration (Figs 1C and S1C). qPCR tests confirmed significant increases in the expression of several representative genes, such as Ripk1, Il6, Stat3, and Ccl2 that were found to be enriched in the gene sets in GSEA analysis (Fig 1D, top panel).
Discussion
The HPA axis plays an essential role in the regulation of body homeostasis. As a major component of the HPA axis, the pituitary has been extensively studied in the context of stress responses and immune challenges. Previous studies have focused on corticotropes in the pituitary, which receive CRH signals from the PVN in the hypothalamus and release the peptide hormone ACTH. The release of ACTH leads to an increase in the serum concentrations of glucocorticoid, which activates the glucocorticoid receptor to control inflammatory gene expression and ultimately suppresses inflammation. This mechanism has served as the basis for the treatment of various clinical diseases, such as acute inflammatory syndromes related to cytokine storm following SARS-CoV-2 infection [61]. However, it remains unknown how systematic inflammation affects the transcriptomic profiles of pituitary cells. Here, we used scRNA-seq and other omics techniques to profile the transcriptional landscape of pituitary populations under healthy and inflammatory conditions. Our data reveal wide-ranging and cell type-specific responses of pituitary HPCs to systemic inflammation and have several functional implications on the pituitary regulations of body physiology.
Acknowledgments
The authors thank all members of ML’s laboratory for their assistance in this study. We thank Fuchou Tan and Yueli Cui (Peking University) for their assistance in scRNA-seq experiments. We thank Chenxi Jia and Di Yao (National Center for Protein Sciences-Beijing) for their assistance in the pituitary proteomics experiments. We thank Tao Cai (National Institute of Biological Sciences, Beijing) for his assistance in bioinformatics analysis. We thank Rui Lin (National Institute of Biological Sciences, Beijing), Xiangyu Li (currently School of Software Engineering, Beijing Jiaotong University), and Hongjun Li (Michael Q. Zhang Lab, TNLIST Bioinformatics Division, Tsinghua University) for discussions.
Citation: Yan T, Wang R, Yao J, Luo M (2023) Single-cell transcriptomic analysis reveals rich pituitary–Immune interactions under systemic inflammation. PLoS Biol 21(12): e3002403. https://doi.org/10.1371/journal.pbio.3002403
Academic Editor: Richard Daneman, UCSD, UNITED STATES
Received: March 14, 2023; Accepted: October 26, 2023; Published: December 18, 2023
Copyright: © 2023 Yan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data generated or analyzed during this study are included in this published article (and its supporting information files). All fastq files, expression matrix and metadata table are available from the National Genomics Data Center (NGDC) under the accession number PRJCA015861. The pipeline for processing the STRT-seq2 data was written in Snakemake and deposited at https://github.com/RuiyuRayWang/ScRNAseq_smkpipe_at_Luolab. Additional codes for analyzing the data were deposited at https://github.com/RuiyuRayWang/pituitary_inflammation.
Funding: This work was supported by Ministry of Science and Technology STI2030-Major Projects (2021ZD0202803), the Research Unit of Medical Neurobiology at Chinese Academy of Medical Sciences (2019RU003), Beijing Municipal Government, Tsinghua University and New Cornerstone Science Foundation to M.L. The funder had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: AAV, adeno-associated virus; BMDM, bone marrow-derived macrophage; CLP, cecal ligation and puncture; CRH, corticotropin-releasing hormone; DEG, differentially expressed gene; DPBS, Dulbecco’s phosphate-buffered saline; FA, formic acid; FACS, fluorescence-activated cell sorting; FSH, follicle-stimulating hormone; GH, growth hormone; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; HPA, hypothalamus–pituitary–adrenal; HPC, hormone-producing cell; HS, horse serum; HVG, highly variable gene; ISH, in situ hybridization; LH, luteinizing hormone; LPS, lipopolysaccharide; MAD, median absolute deviation; M-CSF, macrophage colony-stimulating factor; MSH, melanocyte-stimulating hormone; MWCO, molecular weight cut-off; NBR, neutrophil-to-B cell ratio; NLR, neutrophil-to-lymphocyte ratio; PCA, principal component analysis; PBMC, peripheral blood mononuclear cell; PRL, prolactin; PVN, paraventricular nucleus; RBC, red blood cell; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SCENIC, single-cell regulatory network inference and clustering; scRNA-seq, single-cell RNA-sequencing; STRT-seq, single-cell tagged reverse transcription sequencing; TNF-α, tumor necrosis factor α; TSH, thyroid-stimulating hormone; UMAP, uniform manifold approximation and projection; UMI, unique molecular identifier; WBC, white blood cell
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002403#abstract0
