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Single-cell transcriptomic analysis reveals rich pituitary–Immune interactions under systemic inflammation [1]

['Ting Yan', 'School Of Life Sciences', 'Tsinghua-Peking Center For Life Sciences', 'Tsinghua University', 'Beijing', 'Chinese Institute For Brain Research', 'National Institute Of Biological Sciences', 'Nibs', 'Ruiyu Wang', 'Ptn Graduate Program']

Date: 2024-01

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.

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.

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 .

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.

The emergence of the single-cell RNA sequencing (scRNA-seq) technique has enabled comprehensive characterization of pituitary transcriptional responses in a cell type-specific manner. While prior studies have illuminated the pituitary’s transcriptional landscape in physiological conditions [ 13 – 16 ], systematic investigation into pituitary HPCs’ transcriptomics under systemic inflammatory challenges remains absent. In this study, we performed scRNA-seq of the pituitary glands of mice subjected to systemic immune stimuli. Our findings unveil robust transcriptional responses across all pituitary HPCs in the face of inflammatory challenges, with corticotropes exhibiting the most pronounced reaction. Systemic inflammation potently induces the pituitary expression of many genes encoding secreted factors like cytokines. This approach also led to the identification of Nptx2, a secreted protein from corticotropes, serving as a pituitary inflammation biomarker. Combining cell–cell communication analysis of the transcriptomics with experimental validations revealed extensive crosstalk between pituitary cells and immune cells during inflammation. Together, these results reveal previously underappreciated interactions between the pituitary gland and immune system under inflammatory conditions, highlighting potent effects of immune challenges on a range of HPA-related, fundamental physiological responses.

Several unresolved questions pertain to interactions between the pituitary and the immune system. The pituitary gland comprises 6 major types of hormone-producing cells (HPCs): somatotropes (producing the growth hormones-GH), corticotropes (producing ACTH), lactotropes (producing prolactin-PRL), gonadotropes (producing luteinizing hormone-LH and follicle-stimulating hormone-FSH), thyrotropes (producing thyroid-stimulating hormone-TSH), and melanotropes (producing melanocyte-stimulating hormone-MSH) [ 12 ]. The specific responses of diverse pituitary HPCs to inflammatory stimuli remain uncertain. Clarifying this issue could shed light on how immune stress influences the hormonal regulation of various physiological functions. Additionally, aside from conventional hormones, pituitary HPCs might secrete other factors. Identifying these factors could expand our understanding of HPC functions. Lastly, it remains unclear how direct pituitary–immune communication impacts pituitary physiology.

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 ].

Results

Crosstalks between the pituitary and immune cells during systemic inflammation Our findings thus far suggest that pituitary HPCs respond profoundly to systemic inflammation and may release cytokines as well as other novel immunomodulators to affect the immune system (Figs 3B and S7D). To probe the potential bidirectional endocrine communications between the pituitary and the immune system, we performed cell–cell communication analysis with CellChat [54] using our pituitary scRNA-seq dataset and a previously published scRNA-seq dataset on the spleen [55]. Systemic inflammation was associated with more and overall stronger interactions within and between the pituitary and spleen populations (Fig 4A). More specifically, during systemic inflammation putative signaling increased between the pituitary and spleen populations and within the spleen, whereas it decreased within the pituitary (Fig 4B). These data suggest that inflammation increases the probability of endocrine signaling between the pituitary and the spleen. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Cell–cell communications between the pituitary and the immune cells under healthy or inflammatory states. (A) Bar graph showing the total number (left) and strength (right) of possible interactions between pituitary cells and spleen cells under healthy or inflammatory state by CellChat analysis. (B) Circle plots showing the number of inferred interactions within and between pituitary and spleen populations. Red line: the interactions from the pituitary to the spleen; blue line: the interactions from the spleen to the pituitary. (C) Circle plots showing the number of statistically significant interactions for the CXCL pathways between HPCs in the pituitary and immune cells in the spleen. Each color represents 1 cell type; edges connecting circles represent significant intercellular signaling inferred between those cell types. (D) Inferred interactions from corticotropes to splenic immune cell populations under healthy or inflammatory states. The dot color and size represent the calculated communication probability and p-values; p-values are computed from one-sided permutation test. (E) Transwell index of monocyte, neutrophil, and macrophage recruited by PBS- or LPS-treated AtT-20 cells for 6 h (n = 3 replicates). (F and G) Flow cytometry analysis of macrophages (F) and neutrophils (G) in the pituitary from mice treated with saline or LPS (0.5 mg/kg; n = 3 mice). All data represent mean with SD. Statistical significance was determined by two-tailed Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. The data underlying this figure can be found in S1 Data. HPC, hormone-producing cell; LPS, lipopolysaccharide; NK, natural killer cell; Macro, macrophage; Neut, neutrophil; pDCs, plasmacytoid dendritic cells; Mono, monocyte; T, T cell; B, B cell; Som, somatotropes; Lac, lactotropes; Cort, corticotropes; Mel, melanotropes; Gonad, gonadotropes; Thyro, thyrotropes. https://doi.org/10.1371/journal.pbio.3002403.g004 Next, we explored the communication between pituitary HPCs and spleen immune cells in a cell type- and signaling pathway-specific manner. Systemic inflammation drastically increased the information flow in CXCL signaling between immune cells, especially neutrophils, and all major types of HPCs (Fig 4C). Corticotropes, the prominent responders to inflammation in HPCs, mediated the enhanced connections in CCL and CXCL signaling interactions with many immune cells, especially monocytes and neutrophils (Fig 4D). These results suggest that systemic inflammation promotes the cell–cell signaling between the pituitary cells and immune cell populations in a signaling pathway-specific manner. Given the convenience of studying pituitary–immune cell–cell communications in vitro, we chose to functionally validate the communications using AtT-20 cells. Treating AtT-20 with the serum from LPS-challenged mice or with one of 5 inflammatory stimuli (LPS, IL-1α, IL-1β, TNF-α, and IL-6) significantly up-regulated the expression levels of multiple chemokine-encoding genes (S9A and S9B Fig), demonstrating that inflammatory cytokines could directly modulate the gene expression of corticotropes. Using Transwell migration assay, we found that after being treated with LPS, AtT-20 cells attracted neutrophils, monocytes, and bone marrow-derived macrophages (BMDMs) (Fig 4E), indicating that immune cells can be recruited by inflammatory corticotropes during inflammation in vitro. At the in vivo level, fluorescence-activated cell sorting (FACS) results showed that the number of macrophages (CD45hiCD11b+) [56] increased in the pituitary and returned to normal levels 48 h after LPS treatment, which were consistent with the macrophage marker gene expression level (Figs 4F, S9C–S9E, and S9H). The number of CD206+ M2-like macrophages, a population responsible for tissue repair [57,58], increased 24 h after LPS treatment and peaked at 48 h (S9I–S9L Fig). We also observed dynamic changes in the number of neutrophils in the pituitary (Figs 4G, S9C, S9F, and S9G). Taken together, these results corroborated the findings of the CellChat analysis and suggested that the pituitary is modulated by systemic inflammation to recruit immune cells into the pituitary likely through enhanced chemokine signaling.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002403

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