Circadian clock disruption impairs immune oscillation in chronic endogenous hypercortisolism: a multi-level analysis from a multicentre clinical trialResearch in context
Valeria Hasenmajer,
Emilia Sbardella,
Francesca Sciarra,
Chiara Simeoli,
Claudia Pivonello,
Filippo Ceccato,
Riccardo Pofi,
Marianna Minnetti,
Flavio Rizzo,
Davide Ferrari,
Ilaria Bonaventura,
Federica Barbagallo,
Elisa Giannetta,
Danilo Alunni Fegatelli,
Simone Conia,
Roberto Navigli,
Giorgio Arnaldi,
Carla Scaroni,
Rosario Pivonello,
Daniele Gianfrilli,
Mary Anna Venneri,
Andrea M. Isidori
Affiliations
Valeria Hasenmajer
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Emilia Sbardella
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Francesca Sciarra
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Chiara Simeoli
Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Diabetologia, Andrologia e Nutrizione, Università Federico II di Napoli, Naples, Italy
Claudia Pivonello
Dipartimento di Sanità Pubblica, Università Federico II di Napoli, Naples, Italy
Filippo Ceccato
Endocrinology, Dep of Medicine, DIMED, University-Hospital of Padova, Padua, Italy; Endocrine Disease Unit, University-Hospital of Padova, Padova, Italy
Riccardo Pofi
Oxford Centre for Diabetes, Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre, University of Oxford, Churchill Hospital, Oxford, UK
Marianna Minnetti
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Flavio Rizzo
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Davide Ferrari
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Ilaria Bonaventura
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Federica Barbagallo
Department of Medicine and Surgery, Kore University of Enna, 94100, Enna, Italy
Elisa Giannetta
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Danilo Alunni Fegatelli
Department of Public Health and Infectious Disease, “Sapienza” University of Rome, Rome, Italy
Simone Conia
Sapienza NLP, Department of Computer, Control and Management Engineering, “Sapienza” University of Rome, Rome, Italy
Roberto Navigli
Sapienza NLP, Department of Computer, Control and Management Engineering, “Sapienza” University of Rome, Rome, Italy
Giorgio Arnaldi
Departement of Promozione della Salute, Materno-Infantile, Medicina Interna e Specialistica di Eccellenza “G. D'Alessandro” (PROMISE), Palermo University, 90127, Palermo, Italy
Carla Scaroni
Endocrinology, Dep of Medicine, DIMED, University-Hospital of Padova, Padua, Italy; Endocrine Disease Unit, University-Hospital of Padova, Padova, Italy
Rosario Pivonello
Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Diabetologia, Andrologia e Nutrizione, Università Federico II di Napoli, Naples, Italy
Daniele Gianfrilli
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Mary Anna Venneri
Department of Experimental Medicine, “Sapienza” University of Rome, Italy
Andrea M. Isidori
Department of Experimental Medicine, “Sapienza” University of Rome, Italy; Centre for Rare Diseases (Endo-ERN Accredited), Policlinico Umberto I, Rome, Italy; Corresponding author. Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 329, 00161, Roma, Italy.
Summary: Background: Glucocorticoids (GC) are potent entrainers of the circadian clock. However, their effects on biological rhythms in chronic human exposure have yet to be studied. Endogenous hypercortisolism (Cushing's Syndrome, CS) is a rare condition in which circadian disruption is sustained by a tumorous source of GC excess, offering the unique opportunity to investigate GC's chronic effects in vivo. Methods: In a 12-month prospective case–control multicentre trial, the daily fluctuations in the number of circulating peripheral blood mononuclear cells (PBMCs) and the time-specific expression of clock-related genes were analysed in a cohort of 68 subjects, 34 affected by CS and 34 matched controls. Cosinor mixed effects model, rhythmicity algorithms and machine learning techniques were applied to the multi-level dataset. Findings: Multiple, 5-point daily sampling revealed profound changes in the levels, amplitude, and rhythmicity of several PBMC populations during active CS, only partially restored after remission. Clock gene analyses in isolated PBMCs showed a significant flattening of circadian oscillation of CLOCK, PER1, PER2, PER3, and TIMELESS expression. In active CS, all methods confirmed a loss of rhythmicity of those genes which were circadian in the PBMCs of controls. Most, but not all, genes regained physiological oscillation after remission. Machine learning revealed that while combined time-course sets of clock genes were highly effective in separating patients from controls, immune profiling was efficient even as single time points. Interpretation: In conclusion, the oscillation of circulating immune cells is profoundly altered in patients with CS, representing a convergence point of circadian rhythm disruption and metabolic and steroid hormone imbalances. Machine learning techniques proved the superiority of immune profiling over parameters such as cortisol, anthropometric and metabolic variables, and circadian gene expression analysis to identify CS activity. Funding: The research leading to these results has received funding from the European Union in the context of the National Recovery and Resilience Plan, Investment PE8 – Project Age-It: “Ageing Well in an Ageing Society”. This resource was co-financed by the Next Generation EU [DM 1557 11.10.2022], the PRecisiOn Medicine to Target Frailty of Endocrine-metabolic Origin (PROMETEO) project (NET-2018-12365454) by the Italian Ministry of Health, and through internal funding to Sapienza University of Rome.
