Corrigendum: Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial - the safe iron study.

[This corrects the article DOI: 10.3389/fnut.2023.1230061.].

Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial - the safe iron study.

Introduction: The safety of novel forms of iron in healthy, iron-replete adults as might occur if used in population-based iron supplementation programs was examined. We tested the hypotheses that supplementation with nanoparticulate iron hydroxide adipate tartrate (IHAT), an iron-enriched Aspergillus oryzae product (ASP), or ferrous sulphate heptahydrate (FS) are safe as indicated by erythrocyte susceptibility to malarial infection, bacterial proliferation, and gut inflammation. Responses to FS administered daily or weekly, and with or without other micronutrients were compared. Methods: Two phases of randomized, double-blinded trials were conducted in Boston, MA. Phase I randomized 160 volunteers to six treatments: placebo, IHAT, ASP, FS, and FS plus a micronutrient powder (MNP) administrated daily at 60 mg Fe/day; and FS administered as a single weekly dose of 420 mg Fe. Phase II randomized 86 volunteers to IHAT, ASP, or FS administered at 120 mg Fe/day. Completing these phases were 151 and 77 participants, respectively. The study was powered to detect effects on primary endpoints: susceptibility of participant erythrocytes to infection by Plasmodium falciparum, the proliferation potential of selected pathogenic bacteria in sera, and markers of gut inflammation. Secondary endpoints for which the study was not powered included indicators of iron status and gastrointestinal symptoms. Results: Supplementation with any form of iron did not affect any primary endpoint. In Phase I, the frequency of gastrointestinal symptoms associated with FS was unaffected by dosing with MNP or weekly administration; but participants taking IHAT more frequently reported abdominal pain (27%, p < 0.008) and nausea (4%, p = 0.009) than those taking FS, while those taking ASP more frequently reported nausea (8%, p = 0.009). Surprisingly, only 9% of participants taking IHAT at 120 mg Fe/day (Phase II) reported abdominal pain and no other group reported that symptom. Discussion: With respect to the primary endpoints, few differences were found when comparing these forms of iron, indicating that 28 days of 60 or 120 mg/day of IHAT, ASP, or FS may be safe for healthy, iron-replete adults. With respect to other endpoints, subjects receiving IHAT more frequently reported abdominal pain and nausea, suggesting the need for further study. Clinical Trial Registration: ClinicalTrials.gov, NCT03212677; registered: 11 July 2017.

The Yin and Yang of Pneumolysin During Pneumococcal Infection.

Pneumolysin (PLY) is a pore-forming toxin produced by the human pathobiont Streptococcus pneumoniae, the major cause of pneumonia worldwide. PLY, a key pneumococcal virulence factor, can form transmembrane pores in host cells, disrupting plasma membrane integrity and deregulating cellular homeostasis. At lytic concentrations, PLY causes cell death. At sub-lytic concentrations, PLY triggers host cell survival pathways that cooperate to reseal the damaged plasma membrane and restore cell homeostasis. While PLY is generally considered a pivotal factor promoting S. pneumoniae colonization and survival, it is also a powerful trigger of the innate and adaptive host immune response against bacterial infection. The dichotomy of PLY as both a key bacterial virulence factor and a trigger for host immune modulation allows the toxin to display both "Yin" and "Yang" properties during infection, promoting disease by membrane perforation and activating inflammatory pathways, while also mitigating damage by triggering host cell repair and initiating anti-inflammatory responses. Due to its cytolytic activity and diverse immunomodulatory properties, PLY is integral to every stage of S. pneumoniae pathogenesis and may tip the balance towards either the pathogen or the host depending on the context of infection.

Safe and effective delivery of supplemental iron to healthy older adults: The double-blind, randomized, placebo-controlled trial protocol of the Safe Iron Study.

The forms of iron currently available to correct iron deficiency have adverse effects, including infectious diarrhea, increased susceptibility to malaria, inflammation and detrimental changes to the gut microbiome. These adverse effects limit their use such that the growing burden of iron deficiency has not abated in recent decades. Here, we summarize the protocol of the "Safe Iron Study", the first clinical study examining the safety and efficacy of novel forms of iron in healthy, iron-replete adults. The Safe Iron Study is a double-blind, randomized, placebo-controlled trial conducted in Boston, MA, USA. This study compares ferrous sulfate heptahydrate (FeSO 4·H 2O) with two novel forms of iron supplements (iron hydroxide adipate tartrate (IHAT) and organic fungal iron metabolite (Aspiron™ Natural Koji Iron)). In Phase I, we will compare each source of iron administrated at a low dose (60 mg Fe/day). We will also determine the effect of FeSO 4 co-administrated with a multiple micronutrient powder and weekly administration of FeSO 4. The forms of iron found to produce no adverse effects, or adverse effects no greater than FeSO 4 in Phase I, Phase II will evaluate a higher, i.e., a therapeutic dose (120 mg Fe/day). The primary outcomes of this study include ex vivo malaria ( Plasmodium falciparum) infectivity of host erythrocytes, ex vivo bacterial proliferation (of selected species) in presence of host plasma and intestinal inflammation assessed by fecal calprotectin. This study will test the hypotheses that the novel forms of iron, administered at equivalent doses to FeSO 4, will produce similar increases in iron status in iron-replete subjects, yet lower increases in ex vivo malaria infectivity, ex vivo bacterial proliferation, gut inflammation. Ultimately, this study seeks to contribute to development of safe and effective forms of supplemental iron to address the global burden of iron deficiency and anemia. Registration: ClinicalTrials.gov identifier: NCT03212677; registered: 11 July 2017.

Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A3 (HXA3) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A2 (PLA2) promotes the release of AA, we investigated the role of PLA2 in local and systemic disease during S. pneumoniae infection. The group IVA cytosolic isoform of PLA2 (cPLA2α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae-induced PMN transepithelial migration in vitro Genetic ablation of the cPLA2 isoform cPLA2α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA2α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLA2α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniae lung challenge.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae.

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22-35 years) or elderly (65-69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.

The α-tocopherol form of vitamin E reverses age-associated susceptibility to streptococcus pneumoniae lung infection by modulating pulmonary neutrophil recruitment.

Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22-24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infection. This α-Toc-induced resistance to pneumococcal challenge was associated with a 2-fold fewer pulmonary neutrophils, a level comparable to S. pneumoniae-challenged, conventionally fed young mice. α-Toc directly inhibited neutrophil egress across epithelial cell monolayers in vitro in response to pneumococci or hepoxilin-A3, an eicosanoid required for pneumococcus-elicited neutrophil trans-epithelial migration. α-Toc altered expression of multiple epithelial and neutrophil adhesion molecules involved in migration, including CD55, CD47, CD18/CD11b, and ICAM-1. These findings suggest that α-Toc enhances resistance of aged mice to bacterial pneumonia by modulating the innate immune response, a finding that has potential clinical significance in combating infection in aged individuals through nutritional intervention.

Systemic disease during Streptococcus pneumoniae acute lung infection requires 12-lipoxygenase-dependent inflammation.

Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs), and a risk of systemic spread of the bacterium. We investigated host mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-LOX in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12-LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-LOX exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high-level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX-dependent HXA3 production and subsequent PMN transepithelial migration.

Targeting type III secretion in Yersinia pestis.

Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.