Manfred Nairz

Due to its advantageous redox properties, iron plays an important role in the metabolism of nearly all life. However, these properties are not only a boon but also the bane of such life forms. Since labile iron results in the generation of reactive oxygen species by Fenton chemistry, iron is stored in a relatively safe form inside of ferritin. Despite the fact that the iron storage protein ferritin has been extensively researched, many of its physiological functions are hitherto unresolved. However, research regarding ferritin’s functions is gaining momentum. For example, recent major discoveries on its secretion and distribution mechanisms have been made as well as the paradigm-changing finding of intracellular compartmentalization of ferritin via interaction with nuclear receptor coactivator 4 (NCOA4). In this review, we discuss established knowledge as well as these new findings and the implications they may have for host–pathogen interaction during bacterial infection.

Reverse transcription polymerase chain reaction (RT-PCR) on respiratory tract swabs has become the gold standard for sensitive and specific detection of influenza virus, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this retrospective analysis, we report on the successive implementation and routine use of multiplex RT-PCR testing for patients admitted to the Internal Medicine Emergency Department (ED) at a tertiary care center in Western Austria, one of the hotspots in the early coronavirus disease 2019 (COVID-19) pandemic in Europe. Our description focuses on the use of the Cepheid^® Xpert^® Xpress closed RT-PCR system in point-of-care testing (POCT). Our indications for RT-PCR testing changed during the observation period: From the cold season 2016/2017 until the cold season 2019/2020, we used RT-PCR to diagnose influenza or RSV infection in patients with fever and/or respiratory symptoms. Starting in March 2020, we used the RT-PCR for SARS-CoV-2 and a multiplex version for the combined detection of all these three respiratory viruses to also screen subjects who did not present with symptoms of infection but needed in-hospital medical treatment for other reasons. Expectedly, the switch to a more liberal RT-PCR test strategy resulted in a substantial increase in the number of tests. Nevertheless, we observed an immediate decline in influenza virus and RSV detections in early 2020 that coincided with public SARS-CoV-2 containment measures. In contrast, the extensive use of the combined RT-PCR test enabled us to monitor the re-emergence of influenza and RSV detections, including asymptomatic cases, at the end of 2022 when COVID-19 containment measures were no longer in place. Our analysis of PCR results for respiratory viruses from a real-life setting at an ED provides valuable information on the epidemiology of those infections over several years, their contribution to morbidity and need for hospital admission, the risk for nosocomial introduction of such infection into hospitals from asymptomatic carriers, and guidance as to how general precautions and prophylactic strategies affect the dynamics of those infections.

The sequestration of iron in case of infection, termed nutritional immunity, is an established strategy of host defense. However, the interaction between pathogens and the mammalian iron storage protein ferritin is hitherto not completely understood. To better characterize the function of ferritin in Gram-negative infections, we incubated iron-starved cultures of Salmonella Typhimurium and knockout mutant strains defective for major iron uptake pathways or Escherichia coli with horse spleen ferritin or ionic iron as the sole iron source. Additionally, we added bovine superoxide dismutase and protease inhibitors to the growth medium to assess the effect of superoxide and bacterial proteases, respectively, on Salmonella proliferation and reductive iron release. Compared to free ionic iron, ferritin-bound iron was less available to Salmonella, but was still sufficient to significantly enhance the growth of the bacteria. In the absence of various iron acquisition genes, the availability of ferritin iron further decreased. Supplementation with superoxide dismutase significantly reduced the growth of the ΔentC knockout strain with holoferritin as the sole iron source in comparison with ionic ferrous iron. In contrast, this difference was not observed in the wildtype strain, suggesting that superoxide dismutase undermines bacterial iron uptake from ferritin by siderophore-independent mechanisms. Ferritin seems to diminish iron availability for bacteria in comparison to ionic iron, and its iron sequestering effect could possibly be enhanced by host superoxide dismutase activity.

Coronavirus disease 2019 (COVID-19) is frequently associated with iron dyshomeostasis. The latter is related to acute disease severity and COVID-19 convalescence. We herein describe iron dyshomeostasis at COVID-19 follow-up and its association with long-term pulmonary and symptomatic recovery. The prospective, multicentre, observational cohort study “Development of Interstitial Lung Disease (ILD) in Patients With Severe SARS-CoV-2 Infection (CovILD)” encompasses serial extensive clinical, laboratory, functional and imaging evaluations at 60, 100, 180 and 360 days after COVID-19 onset. We included 108 individuals with mild-to-critical acute COVID-19, whereas 75% presented with severe acute disease. At 60 days post-COVID-19 follow-up, hyperferritinaemia (35% of patients), iron deficiency (24% of the cohort) and anaemia (9% of the patients) were frequently found. Anaemia of inflammation (AI) was the predominant feature at early post-acute follow-up, whereas the anaemia phenotype shifted towards iron deficiency anaemia (IDA) and combinations of IDA and AI until the 360 days follow-up. The prevalence of anaemia significantly decreased over time, but iron dyshomeostasis remained a frequent finding throughout the study. Neither iron dyshomeostasis nor anaemia were related to persisting structural lung impairment, but both were associated with impaired stress resilience at long-term COVID-19 follow-up. To conclude, iron dyshomeostasis and anaemia are frequent findings after COVID-19 and may contribute to its long-term symptomatic outcome.

Background: Pneumonia is often elicited by bacteria and can be associated with a severe clinical course, respiratory failure and the need for mechanical ventilation. In the alveolus, type-2-alveolar-epithelial-cells (AECII) contribute to innate immune functions. We hypothesized that AECII actively adapt cellular iron homeostasis to restrict this essential nutrient from invading pathogens – a defense strategy termed ‘nutritional immunity’, hitherto mainly demonstrated for myeloid cells.Methods: We established an in-vitro infection model using the human AECII-like cell line A549. We infected cells with Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), two gram-negative bacteria with different modes of infection and frequent causes of hospital-acquired pneumonia. We followed the entry and intracellular growth of these gram-negative bacteria and analyzed differential gene expression and protein levels of key inflammatory and iron metabolism molecules.Results: Both, K. pneumoniae and E. coli are able to invade A549 cells, whereas only K. pneumoniae is capable of proliferating intracellularly. After peak bacterial burden, the number of intracellular pathogens declines, suggesting that epithelial cells initiate antimicrobial immune effector pathways to combat bacterial proliferation. The extracellular pathogen E. coli induces an iron retention phenotype in A549 cells, mainly characterized by the downregulation of the pivotal iron exporter ferroportin, the upregulation of the iron importer transferrin-receptor-1 and corresponding induction of the iron storage protein ferritin. In contrast, cells infected with the facultative intracellular bacterium K. pneumoniae exhibit an iron export phenotype indicated by ferroportin upregulation. This differential regulation of iron homeostasis and the pathogen-specific inflammatory reaction is likely mediated by oxidative stress.Conclusion: AECII-derived A549 cells show pathogen-specific innate immune functions and adapt their iron handling in response to infection. The differential regulation of iron transporters depends on the preferential intra- or extracellular localization of the pathogen and likely aims at limiting bacterial iron availability.

Cancer-induced anemia (CIA) is a common consequence of neoplasia and has a multifactorial pathophysiology. The immune response and tumor treatment, both intended to primarily target malignant cells, also affect erythropoiesis in the bone marrow. In parallel, immune activation inevitably induces the iron-regulatory hormone hepcidin to direct iron fluxes away from erythroid progenitors and into compartments of the mononuclear phagocyte system. Moreover, many inflammatory mediators inhibit the synthesis of erythropoietin, which is essential for stimulation and differentiation of erythroid progenitor cells to mature cells ready for release into the blood stream. These pathophysiological hallmarks of CIA imply that the bone marrow is not only deprived of iron as nutrient but also of erythropoietin as central growth factor for erythropoiesis. Tumor-associated macrophages (TAM) are present in the tumor microenvironment and display altered immune and iron phenotypes. On the one hand, their functions are altered by adjacent tumor cells so that they promote rather than inhibit the growth of malignant cells. As consequences, TAM may deliver iron to tumor cells and produce reduced amounts of cytotoxic mediators. Furthermore, their ability to stimulate adaptive anti-tumor immune responses is severely compromised. On the other hand, TAM are potential off-targets of therapeutic interventions against CIA. Red blood cell transfusions, intravenous iron preparations, erythropoiesis-stimulating agents and novel treatment options for CIA may interfere with TAM function and thus exhibit secondary effects on the underlying malignancy. In this Hypothesis and Theory, we summarize the pathophysiological hallmarks, clinical implications and treatment strategies for CIA. Focusing on TAM, we speculate on the potential intended and unintended effects that therapeutic options for CIA may have on the innate immune response and, consequently, on the course of the underlying malignancy.

Genetic and dietary forms of iron overload have distinctive clinical and pathophysiological features. HFE-associated hereditary hemochromatosis is characterized by overwhelming intestinal iron absorption, parenchymal iron deposition and macrophage iron depletion. In contrast, excessive dietary iron intake results in iron deposition in macrophages. However, the functional consequences of genetic and dietary iron overload for the control of microbes are incompletely understood. Using Hfe+/+ and Hfe-/- mice in combination with oral iron overload in a model of Salmonella enterica serovar Typhimurium infection, we found animals of either genotype to induce hepcidin antimicrobial peptide expression and hypoferremia following systemic infection in an Hfe-independent manner. As predicted, Hfe-/- mice, a model of hereditary hemochromatosis, displayed reduced spleen iron content, which translated into improved control of Salmonella replication. Salmonella adapted to the iron-poor microenvironment in the spleens of Hfe-/- mice by inducing the expression of its siderophore iron-uptake machinery. Dietary iron loading resulted in higher bacterial numbers in both WT and Hfe-/- mice, although Hfe deficiency still resulted in better pathogen control and improved survival. This suggests that Hfe deficiency may exert protective effects in addition to the control of iron availability for intracellular bacteria. Our data show that a dynamic adaptation of iron metabolism in both immune cells and microbes shapes the host-pathogen interaction in the setting of systemic Salmonella infection. Moreover, Hfe-associated iron overload and dietary iron excess result in different outcomes in infection, indicating that tissue and cellular iron distribution determines the susceptibility to infection with specific pathogens.