key: cord-0741394-unpy75qf
authors: Poma, Anello Marcello; Proietti, Agnese; Macerola, Elisabetta; Bonuccelli, Diana; Conti, Marco; Salvetti, Alessandra; Dolo, Vincenza; Chillà, Andrea; Basolo, Alessio; Santini, Ferruccio; Toniolo, Antonio; Basolo, Fulvio
title: Suppression of pituitary hormone genes in subjects who died from COVID-19 independently of virus detection in the gland
date: 2022-05-14
journal: J Clin Endocrinol Metab
DOI: 10.1210/clinem/dgac312
sha: 5b6a5e10ca89fbe8fe3dc98e4d9f72a2a2c42d20
doc_id: 741394
cord_uid: unpy75qf

CONTEXT: Involvement of the pituitary gland in SARS-CoV-2 infection has been clinically suggested by pituitary hormone deficiency in severe COVID-19 cases, by altered serum ACTH levels in hospitalized patients, and by cases of pituitary apoplexy. However, the direct viral infection of the gland has not been investigated. OBJECTIVES: To evaluate whether the SARS-CoV-2 genome and antigens could be present in pituitary glands of lethal cases of COVID-19, and to assess possible changes in the expression of immune-related and pituitary-specific genes. METHODS: SARS-CoV-2 genome and antigens were searched in the pituitary gland of 23 patients who died from COVID-19 and, as controls, in 12 subjects who died from trauma or sudden cardiac death. Real-time RT-PCR, in situ hybridization, immunohistochemistry and transmission electron microscopy were utilized. Levels of mRNA transcripts of immune-related and pituitary-specific genes were measured by the nCounter assay. RESULTS: The SARS-CoV-2 genome and antigens were detected in 14/23 (61%) pituitary glands of the COVID-19 group, not in controls. In SARS-CoV-2 positive pituitaries, the viral genome was consistently detected by PCR in the adeno- and the neurohypophysis. Immunohistochemistry, in situ hybridization and transmission electron microscopy confirmed the presence of SARS-CoV-2 in the pituitary. Activation of type I interferon signaling and enhanced levels of neutrophil and cytotoxic cell scores were found in virus-positive glands. mRNA transcripts of pituitary hormones and pituitary developmental/regulatory genes were suppressed in all COVID-19 cases irrespective of virus-positivity. CONCLUSION: Our study supports the tropism of SARS-CoV-2 for human pituitary and encourage to explore pituitary dysfunction post-COVID-19.

The Coronavirus Disease-2019 (COVID-19) caused by multiple variants of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been spreading worldwide for over two years provoking deaths and sequelae in the respiratory system and extrapulmonary organs [1] . Ample evidence shows that during the 2003 outbreak of the Severe Acute Respiratory Syndrome (SARS) due to SARS-CoV-1, many patients suffered extrapulmonary sequelae in the gastrointestinal, cardiovascular, immune, nervous, and endocrine systems [2, 3] .

Confirming previous observations on infection by SARS-CoV-1 (which is strictly linked to SARS-CoV-2), even the COVID-19 pandemic may influence endocrine organs such as the adrenals [4, 5] , thyroid [6] [7] [8] [9] , testes [10] [11] [12] , ovaries [13] , pancreatic islets [14] [15] [16] [17] , and pituitary [5, 6, 18, 19] . In lethal cases of COVID-19, autopsy studies revealed the virus within endocrine cells and explored the alterations of endocrine organs [13, 20] . Clinical studies analyzed the endocrine consequences of COVID-19 [2, 3, [21] [22] [23] .

As reported by Frara and colleagues [24] , pre-existing pituitary disorders may aggravate COVID-19.

For instance, Cushing disease and acromegaly predispose to obesity, visceral adiposity, and diabetes mellitus. Untreated growth hormone (GH) deficiency associates with abnormal lipid profile and increased production of proinflammatory cytokines. Hypopituitarism, acromegaly, Cushing disease, adenomas secreting thyroid-stimulating hormone (TSH) are associated with vertebral fractures that aggravate the respiratory dysfunction typical of COVID-19. Also, due to the possible administration of glucocorticoids, hospitalized COVID-19 patients tend to have reduced levels (<30 pg/mL) of adrenocorticotropic hormone (ACTH) while patients with mild to medium severity conditions have ACTH levels higher than normal [19] . Low ACTH levels appear to correlate with disease severity [5] and a dramatic fall of ACTH is a hallmark of severe COVID-19 [19] . In addition, it has been shown that adrenals may be directly infected with SARS-CoV-2 [4] . The resulting cellular damage could predispose COVID-19 patients to adrenal dysfunction [4] . In inflammatory diseases, cytokines tend to enhance ACTH production [25] , but, in severe cases, the feedback acting on the hypothalamus-A c c e p t e d M a n u s c r i p t 4 pituitary-adrenal (HPA) axis is often impaired, and ACTH levels remain low [5] . Impairment of the pituitary-thyroid axis has been documented in the clinics [6] , while central diabetes insipidus (ADH deficiency) has been reported as a consequence of COVID-19 [18] . A few cases of pituitary apoplexy have been reported in COVID-19, both in patients with pituitary adenoma [26] [27] [28] [29] and in patients with conserved pituitary function [30, 31] .

Physiological and pathological conditions may influence the secretion of pituitary hormones, including infection. For these reasons, we investigated autoptic COVID-19 cases who died from SARS-CoV-2 infection to assess whether the virus could be found in the pituitary gland. In several cases, the virus was found in cells of the adeno-and neurohypophysis. Pituitary infection was associated with altered transcription of immune-and hormone-related genes.

All patients were of Caucasian ethnicity and were recruited between November 2020 and January 2022. Three case groups were investigated: a) controls (i.e., subjects who died from acute causes other than infectious, such as trauma and sudden cardiac death; n=12); b) patients who died from COVID-19 that had pituitary tissue negative for SARS-CoV-2 (n=9); patients who died from COVID-19 that had pituitary tissue positive for SARS-CoV-2 (n=14). Patients were treated according to COVID-19 protocols that were standard at the time of hospitalization [32, 33] . Received treatments did not include antivirals or IL1/IL6 blockade [34] . Prior to COVID-19, none of the patients had clinical evidence of pituitary dysfunction.

At autopsy, the pituitary gland was accurately removed, fixed in formalin, and embedded in paraffin for pathological evaluation and for molecular analyses. Tissue samples for transmission electron microscopy were also taken. In 15/23 COVID-19 cases, the neurohypophysis was also sampled. Due to the low amount of material, neurohypophysis tissue was used only for SARS-CoV-2 detection, and gene expression analysis could not be done.

A c c e p t e d M a n u s c r i p t 5 At pathological examination, diffuse alveolar damage, fibrosis, and T-cell infiltration of lung parenchyma was found in all COVID-19 cases, and the pathological report defined COVID-19 as the cause of death. Two lung samples were tested for SARS-CoV-2 genome by real-time RT-PCR. All COVID-19 cases were positive while virus was not detected in controls. In both COVID-19 cases and controls, histopathological examination of pituitaries did not detect neoplastic lesions.

The study was approved by the local Ethics Committee (Comitato Etico Area Vasta Nord-Ovest, Italy; protocol number 17327, 2020-05-14).

For each case, RNA was purified from three 10µm-thick unstained sections using the RNeasy FFPE kit (Qiagen, Hilden, Germany). The quality and quantity of RNA were assessed by spectrophotometry (Trinean, Gentbrugge, Belgium). About 250 ng of RNA were used for the one step real-time RT-PCR assay to detect the SARS-CoV-2 genome in lung and pituitary specimens. The Easy SARS-CoV-2 WE kit (Diatech Pharmacogenetics, Jesi, Italy), validated for in vitro diagnostics, was used on a Rotor-Gene Q instrument (Qiagen). The assay has a limit of detection of 5 target copies per reaction. The assay steps were as follows: retro-transcription at 50°C for 10 minutes; activation of Taq polymerase at 95°C for 5 minutes; 40 cycles of denaturation at 95°C for 5 seconds and annealing/extension at 58°C for 30 seconds. The amplification of an internal control (VPS29) ensured the sample adequacy in terms of amplifiability. A sample was deemed positive when at least one of the two target genes [i.e., nucleocapsid (N) and RNA-dependent RNA polymerase (RdRp) genes] was amplified before the 36 th Ct (N gene) and before the 38 th Ct (RdRp gene).

In situ hybridization (ISH) for SARS-CoV-2 RNA was performed using the RNAscope Probe V- Four µm-thick FFPE sections were used; tissue retrieval was performed using specific Target Retrieval Reagents at 100°C for 15 minutes. After washing steps, treatment with proteases was performed at 40°C for 30 minutes. Then, a series of signal amplification steps by hybridization were performed. Signal detection was carried out by DAB staining. Slides were counterstained with hematoxylin.

Gene expression assay was performed using the nCounter system (nanoString Technologies, Seattle, Protein Atlas (https://www.proteinatlas.org/). The top genes that were reported as tissue-enriched in the human pituitary were selected, and genes that were enriched also in other tissues or organs were filtered out. The complete list of the analyzed transcripts is reported in Supplementary Table S1 [35] . 

Small blocks of adenohypophysis of approximately 1 mm 3 were fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer for 2-4 hours at 4°C. Samples were then processed as described by Cassella and colleagues [36] . Briefly, after post-fixation in 1% osmium tetroxide and dehydration in a graded series of ethanol, they were embedded in an "Epon-Araldite" resin. Ultrathin sections were stained with uranyl acetate and lead citrate and analysed with a Jeol 100 SX transmission electron microscope. Digital images and measurements were acquired using AMT image capture software.

Raw gene expression counts were normalized using the Advanced Analysis module of the nSolver Signatures Database (MutSigDB) v.7.4 were used as reference [37] , and a minimum gene set size of 20 genes was set. Pathway enrichment analysis was performed following the procedures of clusterProfiler Bioconductor package and using the Kyoto encyclopedia of genes and genomes A c c e p t e d M a n u s c r i p t 8 (KEGG) database as reference. Immune cell scores were calculated using the method described by Middleton [38] ; only immune cell scores with at least two markers were considered. The Dunn test for multiple comparisons was used to evaluate differences in terms of immune cell scores and infiltrates. All analyses were performed in R environment v.4.1.2 (https://www.r-project.org/, last accessed January 28, 2022) unless otherwise specified. Table 1 summarizes the demographic and clinical data of controls and COVID-19 cases. Cases of the COVID-19 cohort were older than controls and more frequently obese or overweight. Comorbidities were highly prevalent in both cohorts, with cardiovascular disease being the most frequent. RNA quality was suitable for further analyses, with A260/A280 ratio of 1. [44] .

Results and gene functions are summarized in Table 2 

Multiple extrapulmonary manifestations of COVID-19 have been described [1] . Following SARS-CoV-2 infection, endocrine defects may become apparent during the acute or the post-acute phases [22] .

Previous autopsy studies by our group and others showed that SARS-CoV-2 antigens and genome were present in the thyroid, testis and subcutaneous adipose tissue of individuals who died of COVID-19 [13, 20, 45, 46] . Evidence from the above studies also showed that virus infection of thyroid promotes activation of interferon signalling and immune changes consistent with sub-acute thyroiditis [45] . In COVID-19, additional endocrine dysfunctions have been reported, and post-COVID-19 monitoring is granted in order to administer suitable therapies.

Case reports of pituitary apoplexy in the course of COVID-19 have been documented [26] [27] [28] [29] .

Besides acute events in patients with pre-existing conditions, hypopituitarism has been hypothesized in severe cases [5, 19] A c c e p t e d M a n u s c r i p t 12 small but consistent down-regulation of immune-related genes. Transcription levels of pituitaryspecific genes were also measured. As compared to controls, transcription of the FSHB, TSHB and LHB genes was strongly suppressed in pituitary glands infected by SARS-CoV-2. In addition, the mRNA levels of LHX3 -a transcription factor that regulates pituitary development and the transcription of pituitary-specific genes [47, 48] -were also downregulated. Heterozygous germline mutations in LHX3 gene determine a combined deficiency of pituitary hormones [49, 50] . Regarding [51] [52] [53] . Consistently with our previous findings in autoptic thyroid and adipose tissues [45, 46] , the type I interferon pathway was selectively activated in pituitary glands that were positive for both genome and antigens of SARS-CoV-2. Finally, we observed higher levels of cytotoxic cell and neutrophil scores in virus-positive pituitary of COVID-19 cases. However, focal immune/inflammatory infiltrates were present only in a part of the cases.

Our study has some limitations. First, the number of investigated cases is relatively small due to the risky procedure for collecting the pituitary from sella turcica of infectious COVID-19 cases. Second, immunostaining for CD15 and CD68 markers was not satisfactory for assessing infiltrates of granulocytes and macrophages due to the diffuse expression of these markers in pituitary cells.

Additionally, only a few cases presented CD8 and CD3 staining, thus not providing sufficient data for statistical comparisons. All subjects were of Caucasian ethnicity which hindered the ability of modelling ethnical factors. Finally, hormone dosage was not available since it is not a routine practice in hospitalized COVID-19 patients; hence we could not establish whether SARS-CoV-2 infection was associated with any degree of hypopituitarism.

A c c e p t e d M a n u s c r i p t 13 In closing, the study shows that SARS-CoV-2 infection of the pituitary gland may occur at least in severe forms of COVID-19. As in other endocrine tissues [45, 46] , the infection activates the type I interferon response and appears to reduce the transcription of pituitary-specific hormones irrespective of the direct viral infection of pituitary cells ( Figure 6 ). While COVID-19 infections are more frequently associated with a severe course and higher mortality in men, women appear more predisposed to long COVID [54] . Clinicians should be aware of sexual dimorphism in COVID-19 and the differential long-term consequences of this pandemic virus. Thus, they are advised to follow-up COVID-19 patients for signs of pituitary disorders. As for cardiovascular pathology [55] , studies of long-term endocrine outcomes of COVID-19 will better define the wide pathogenic spectrum of SARS-CoV-2 in humans. M a n u s c r i p t 26 A c c e p t e d M a n u s c r i p t 32 Figure 6 

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A c c e p t e d M a n u s c r i p t 25