Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 μM, cross-linked calprotectin (5 μM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 μM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9) 2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.

Background In Canterbury, near complete identification of coronavirus disease 2019 (COVID‐19) cases during a limited outbreak provides unique insights into sequelae. Aims The current study aimed to measure symptom persistence, time to return to normal activity, generalised anxiety and health‐related quality of life (HrQoL) among COVID‐19 survivors compared with uninfected participants. Methods The authors conducted a prospective cohort study of people tested for COVID‐19 by reverse transcriptase polymerase chain reaction of nasopharyngeal swabs from 1 March to 30 June 2020. They enrolled participants who tested positive and negative at a 1:2 ratio, and administered community‐acquired pneumonia, 7‐item generalised anxiety disorder (GAD‐7) and HrQoL (RAND‐36) questionnaires. Results The authors recruited 145 participants, 48 with COVID‐19 and 97 without COVID‐19. The mean time from COVID‐19 testing to completing the health questionnaire was 306 days. The mean age of patients was 46.7 years, and 70% were women. Four (8%) COVID‐19–positive and eight (8%) COVID‐19–negative participants required hospitalisation. Fatigue (30/48 [63%] vs 13/97 [13%]; P < 0.001), dyspnoea (13/48 [27%] vs 6/97 [6%]; P < 0.001) and chest pain (10/48 [21%] vs 1/97 [1%]; P < 0.001) were persistent in those with COVID‐19. Fewer COVID‐19–positive participants returned to normal activity levels (35/48 [73%] vs 94/97 97%; P < 0.001), with longer times taken (median 21 vs 14 days; P = 0.007). The GAD‐7 and RAND‐36 scores of both groups were similar across all anxiety and HrQoL subscales. Conclusions Persistent symptoms and longer recovery times were found in COVID‐19 survivors, but not impaired generalised anxiety levels or HrQoL compared with COVID‐19–uninfected participants.

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