Hormesis as an adaptive response to infection.

Hormesis is a phenomenon whereby low-level stress can improve cellular, organ, or organismal fitness in response to a subsequent similar or other stress insult. Whereas hormesis is thought to contribute to the fitness benefits arising from symbiotic host-microbe interactions, the putative benefits of hormesis in host-pathogen interactions have yet to be explored. Hormetic responses have nonetheless been reported in experimental models of infection, a common feature of which is regulation of host mitochondrial function. We propose that these mitohormetic responses could be harnessed therapeutically to limit the severity of infectious diseases.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Reframing sepsis immunobiology for translation: towards informative subtyping and targeted immunomodulatory therapies.

Sepsis is a common and deadly condition. Within the current model of sepsis immunobiology, the framing of dysregulated host immune responses into proinflammatory and immunosuppressive responses for the testing of novel treatments has not resulted in successful immunomodulatory therapies. Thus, the recent focus has been to parse observable heterogeneity into subtypes of sepsis to enable personalised immunomodulation. In this Personal View, we highlight that many fundamental immunological concepts such as resistance, disease tolerance, resilience, resolution, and repair are not incorporated into the current sepsis immunobiology model. The focus for addressing heterogeneity in sepsis should be broadened beyond subtyping to encompass the identification of deterministic molecular networks or dominant mechanisms. We explicitly reframe the dysregulated host immune responses in sepsis as altered homoeostasis with pathological disruption of immune-driven resistance, disease tolerance, resilience, and resolution mechanisms. Our proposal highlights opportunities to identify novel treatment targets and could enable successful immunomodulation in the future.

Targeting circulating labile heme as a defense strategy against malaria.

Severe presentations of malaria emerge as Plasmodium (P.) spp. parasites invade and lyse red blood cells (RBC), producing extracellular hemoglobin (HB), from which labile heme is released. Here, we tested whether scavenging of extracellular HB and/or labile heme, by haptoglobin (HP) and/or hemopexin (HPX), respectively, counter the pathogenesis of severe presentations of malaria. We found that circulating labile heme is an independent risk factor for cerebral and non-cerebral presentations of severe P. falciparum malaria in children. Labile heme was negatively correlated with circulating HP and HPX, which were, however, not risk factors for severe P. falciparum malaria. Genetic Hp and/or Hpx deletion in mice led to labile heme accumulation in plasma and kidneys, upon Plasmodium infection This was associated with higher incidence of mortality and acute kidney injury (AKI) in ageing but not adult Plasmodium-infected mice, and was corroborated by an inverse correlation between heme and HPX with serological markers of AKI in P. falciparum malaria. In conclusion, HP and HPX act in an age-dependent manner to prevent the pathogenesis of severe presentation of malaria in mice and presumably in humans.

Single-cell RNA sequencing and analysis of rodent blood stage Plasmodium.

Bulk RNA sequencing of Plasmodium spp., the causative parasite of malaria, fails to discriminate developmental-stage-specific gene regulation. Here, we provide a protocol that uses single-cell RNA sequencing of FACS-sorted Plasmodium-chabaudi-chabaudi-AS-infected red blood cells (iRBCs) to characterize developmental-stage-specific modulation of gene expression during malaria blood stage. We describe steps for infecting mice, monitoring disease progression, preparing iRBCs, and single-cell sequencing iRBCs. We then detail procedures for analyzing scRNA-seq data. For complete details on the use and execution of this protocol, please refer to Ramos et al.1.

Renal control of life-threatening malarial anemia.

Iron recycling prevents the development of anemia under homeostatic conditions. Whether iron recycling was co-opted as a defense strategy to prevent the development of anemia in response to infection is unclear. We find that in severe Plasmodium falciparum malaria, the onset of life-threatening anemia is associated with acute kidney injury (AKI), irrespective of parasite load. Using a well-established experimental rodent model of malaria anemia, we identify a transcriptional response that endows renal proximal tubule epithelial cells (RPTECs) with the capacity to store and recycle iron during P. chabaudi chabaudi (Pcc) infection. This response encompasses the induction of ferroportin 1/SLC40A1, which exports iron from RPTECs and counteracts AKI while supporting compensatory erythropoiesis and preventing the onset of life-threatening malarial anemia. Iron recycling by myeloid cells is dispensable to this protective response, suggesting that RPTECs provide an iron-recycling salvage pathway that prevents the pathogenesis of life-threatening malarial anemia.

DNA damage independent inhibition of NF-κB transcription by anthracyclines.

Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon-responsive genes in primary mouse (Mus musculus) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.

A hypometabolic defense strategy against malaria.

Hypoglycemia is a clinical hallmark of severe malaria, the often-lethal outcome of Plasmodium falciparum infection. Here, we report that malaria-associated hypoglycemia emerges from a non-canonical resistance mechanism, whereby the infected host reduces glycemia to starve Plasmodium. This hypometabolic response is elicited by labile heme, a byproduct of hemolysis that induces illness-induced anorexia and represses hepatic glucose production. While transient repression of hepatic glucose production prevents unfettered immune-mediated inflammation, organ damage, and anemia, when sustained over time it leads to hypoglycemia, compromising host energy expenditure and adaptive thermoregulation. The latter arrests the development of asexual stages of Plasmodium via a mechanism associated with parasite mitochondrial dysfunction. In response, Plasmodium activates a transcriptional program associated with the reduction of virulence and sexual differentiation toward the generation of transmissible gametocytes. In conclusion, malaria-associated hypoglycemia represents a trade-off of a hypometabolic-based defense strategy that balances parasite virulence versus transmission.

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme.

Trained immunity defines long-lasting adaptations of innate immunity based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors [M. Divangahi et al., Nat. Immunol. 22, 2-6 (2021)]. Innate immune cells, however, do not exclusively differentiate between foreign and self but also react to host-derived molecules referred to as alarmins. Extracellular "labile" heme, released during infections, is a bona fide alarmin promoting myeloid cell activation [M. P. Soares, M. T. Bozza, Curr. Opin. Immunol. 38, 94-100 (2016)]. Here, we report that labile heme is a previously unrecognized inducer of trained immunity that confers long-term regulation of lineage specification of hematopoietic stem cells and progenitor cells. In contrast to previous reports on trained immunity, essentially mediated by pathogen-associated molecular patterns, heme training depends on spleen tyrosine kinase signal transduction pathway acting upstream of c-Jun N-terminal kinases. Heme training promotes resistance to sepsis, is associated with the expansion of self-renewing hematopoetic stem cells primed toward myelopoiesis and to the occurrence of a specific myeloid cell population. This is potentially evoked by sustained activity of Nfix, Runx1, and Nfe2l2 and dissociation of the transcriptional repressor Bach2. Previously reported trained immunity inducers are, however, infrequently present in the host, whereas heme abundantly occurs during noninfectious and infectious disease. This difference might explain the vanishing protection exerted by heme training in sepsis over time with sustained long-term myeloid adaptations. Hence, we propose that trained immunity is an integral component of innate immunity with distinct functional differences on infectious disease outcome depending on its induction by pathogenic or endogenous molecules.

Glycan-based shaping of the microbiota during primate evolution.

Genes encoding glycosyltransferases can be under relatively high selection pressure, likely due to the involvement of the glycans synthesized in host-microbe interactions. Here, we used mice as an experimental model system to investigate whether loss of α-1,3-galactosyltransferase gene (GGTA1) function and Galα1-3Galß1-4GlcNAcß1-R (αGal) glycan expression affects host-microbiota interactions, as might have occurred during primate evolution. We found that Ggta1 deletion shaped the composition of the gut microbiota. This occurred via an immunoglobulin (Ig)-dependent mechanism, associated with targeting of αGal-expressing bacteria by IgA. Systemic infection with an Ig-shaped microbiota inoculum elicited a less severe form of sepsis compared to infection with non-Ig-shaped microbiota. This suggests that in the absence of host αGal, antibodies can shape the microbiota towards lower pathogenicity. Given the fitness cost imposed by bacterial sepsis, we infer that the observed reduction in microbiota pathogenicity upon Ggta1 deletion in mice may have contributed to increase the frequency of GGTA1 loss-of-function mutations in ancestral primates that gave rise to humans.

Loss of α-gal during primate evolution enhanced antibody-effector function and resistance to bacterial sepsis.

Most mammals express a functional GGTA1 gene encoding the N-acetyllactosaminide α-1,3-galactosyltransferase enzyme, which synthesizes Gal-α1-3Gal-ß1-4GlcNAc (α-gal) and are thus tolerant to this self-expressed glycan. Old World primates including humans, however, carry loss-of-function mutations in GGTA1 and lack α-gal. Presumably, fixation of such mutations was propelled by natural selection, favoring the emergence of α-gal-specific immunity, conferring resistance to α-gal-expressing pathogens. Here, we show that loss of Ggta1 function in mice enhances resistance to bacterial sepsis, irrespectively of α-Gal-specific immunity. Rather, the absence of α-gal from IgG-associated glycans increases IgG effector function via a mechanism associated with enhanced IgG-Fc gamma receptor (FcγR) binding. The ensuing survival advantage against sepsis comes alongside a cost of accelerated reproductive senescence in Ggta1-deleted mice. Mathematical modeling of this trade-off suggests that high exposure to virulent pathogens exerts sufficient selective pressure to fix GGTA1 loss-of-function mutations, as likely occurred during the evolution of primates toward humans.

M. tuberculosis Reprograms Hematopoietic Stem Cells to Limit Myelopoiesis and Impair Trained Immunity.

A greater understanding of hematopoietic stem cell (HSC) regulation is required for dissecting protective versus detrimental immunity to pathogens that cause chronic infections such as Mycobacterium tuberculosis (Mtb). We have shown that systemic administration of Bacille Calmette-Guérin (BCG) or ß-glucan reprograms HSCs in the bone marrow (BM) via a type II interferon (IFN-II) or interleukin-1 (IL1) response, respectively, which confers protective trained immunity against Mtb. Here, we demonstrate that, unlike BCG or ß-glucan, Mtb reprograms HSCs via an IFN-I response that suppresses myelopoiesis and impairs development of protective trained immunity to Mtb. Mechanistically, IFN-I signaling dysregulates iron metabolism, depolarizes mitochondrial membrane potential, and induces cell death specifically in myeloid progenitors. Additionally, activation of the IFN-I/iron axis in HSCs impairs trained immunity to Mtb infection. These results identify an unanticipated immune evasion strategy of Mtb in the BM that controls the magnitude and intrinsic anti-microbial capacity of innate immunity to infection.

Heme Oxygenase-1 Induction by Blood-Feeding Arthropods Controls Skin Inflammation and Promotes Disease Tolerance.

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.

Interleukin-1 promotes autoimmune neuroinflammation by suppressing endothelial heme oxygenase-1 at the blood-brain barrier.

The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood-brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.

Donor-Derived Myeloid Heme Oxygenase-1 Controls the Development of Graft-Versus-Host Disease.

Graft-versus-host disease (GVHD) remains a major clinical drawback of allogeneic hematopoietic stem cell transplantation (HSCT). Here, we investigated how the stress responsive heme catabolizing enzyme heme oxygenase-1 (HO-1, encoded by HMOX1) regulates GVHD in response to allogeneic hematopoietic stem cell transplantation in mice and humans. We found that deletion of the Hmox1 allele, specifically in the myeloid compartment of mouse donor bone marrow, promotes the development of aggressive GVHD after allogeneic transplantation. The mechanism driving GVHD in mice transplanted with allogeneic bone marrow lacking HO-1 expression in the myeloid compartment involves enhanced T cell alloreactivity. The clinical relevance of these observations was validated in two independent cohorts of HSCT patients. Individuals transplanted with hematopoietic stem cells from donors carrying a long homozygous (GT)n repeat polymorphism (L/L) in the HMOX1 promoter, which is associated with lower HO-1 expression, were at higher risk of developing severe acute GVHD as compared to donors carrying a short (GT)n repeat (S/L or S/S) polymorphism associated with higher HO-1 expression. In this study, we showed the unique importance of donor-derived myeloid HO-1 in the prevention of lethal experimental GVHD and we corroborated this observation by demonstrating the association between human HMOX1 (GT)n microsatellite polymorphisms and the incidence of severe acute GVHD in two independent HSCT patient cohorts. Donor-derived myeloid HO-1 constitutes a potential therapeutic target for HSCT patients and large-scale prospective studies in HSCT patients are necessary to validate the HO-1 L/L genotype as an independent risk factor for developing severe acute GVHD.

Labile heme impairs hepatic microcirculation and promotes hepatic injury.

Sepsis is a life-threatening clinical syndrome defined as a deregulated host response to infection associated with organ dysfunction. Mechanisms underlying the pathophysiology of septic liver dysfunction are incompletely understood. Among others, the iron containing tetrapyrrole heme inflicts hepatic damage when released into the circulation during systemic inflammation and sepsis. Accordingly, hemolysis and decreased concentrations of heme-scavenging proteins coincide with an unfavorable outcome of critically ill patients. As the liver is a key organ in heme metabolism and host response to infection, we investigated the impact of labile heme on sinusoidal microcirculation and hepatocellular integrity. We here provide experimental evidence that heme increases portal pressure via a mechanism that involves hepatic stellate cell-mediated sinusoidal constriction, a hallmark of microcirculatory failure under stress conditions. Moreover, heme exerts direct cytotoxicity in vitro and aggravates tissue damage in a model of polymicrobial sepsis. Heme binding by albumin, a low-affinity but high-capacity heme scavenger, attenuates heme-mediated vasoconstriction in vivo and prevents heme-mediated cytotoxicity in vitro. We demonstrate that fractions of serum albumin-bound labile heme are increased in septic patients. We propose that heme scavenging might be used therapeutically to maintain hepatic microcirculation and organ function in sepsis.

Ferritin regulates organismal energy balance and thermogenesis.

OBJECTIVE: The ferritin heavy/heart chain (FTH) gene encodes the ferroxidase component of the iron (Fe) sequestering ferritin complex, which plays a central role in the regulation of cellular Fe metabolism. Here we tested the hypothesis that ferritin regulates organismal Fe metabolism in a manner that impacts energy balance and thermal homeostasis. METHODS: We developed a mouse strain, referred herein as FthR26 fl/fl, expressing a tamoxifen-inducible Cre recombinase under the control of the Rosa26 (R26) promoter and carrying two LoxP (fl) sites: one at the 5'end of the Fth promoter and another the 3' end of the first Fth exon. Tamoxifen administration induces global deletion of Fth in adult FthR26Δ/Δ mice, testing whether FTH is required for maintenance of organismal homeostasis. RESULTS: Under standard nutritional Fe supply, Fth deletion in adult FthR26Δ/Δ mice led to a profound deregulation of organismal Fe metabolism, oxidative stress, inflammation, and multi-organ damage, culminating in death. Unexpectedly, Fth deletion was also associated with a profound atrophy of white and brown adipose tissue as well as with collapse of energy expenditure and thermogenesis. This was attributed mechanistically to mitochondrial dysfunction, as assessed in the liver and in adipose tissue. CONCLUSION: The FTH component of ferritin acts as a master regulator of organismal Fe homeostasis, coupling nutritional Fe supply to organismal redox homeostasis, energy expenditure and thermoregulation.

Renal control of disease tolerance to malaria.

Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill Plasmodium However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.

Disease Tolerance as an Inherent Component of Immunity.

Pathogenic organisms exert a negative impact on host health, revealed by the clinical signs of infectious diseases. Immunity limits the severity of infectious diseases through resistance mechanisms that sense and target pathogens for containment, killing, or expulsion. These resistance mechanisms are viewed as the prevailing function of immunity. Under pathophysiologic conditions, however, immunity arises in response to infections that carry health and fitness costs to the host. Therefore, additional defense mechanisms are required to limit these costs, before immunity becomes operational as well as thereafter to avoid immunopathology. These are tissue damage control mechanisms that adjust the metabolic output of host tissues to different forms of stress and damage associated with infection. Disease tolerance is the term used to define this defense strategy, which does not exert a direct impact on pathogens but is essential to limit the health and fitness costs of infection. Under this argument, we propose that disease tolerance is an inherent component of immunity.