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.

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.

Metabolic Adaptation Establishes Disease Tolerance to Sepsis.

Sepsis is an often lethal syndrome resulting from maladaptive immune and metabolic responses to infection, compromising host homeostasis. Disease tolerance is a defense strategy against infection that preserves host homeostasis without exerting a direct negative impact on pathogens. Here, we demonstrate that induction of the iron-sequestering ferritin H chain (FTH) in response to polymicrobial infections is critical to establish disease tolerance to sepsis. The protective effect of FTH is exerted via a mechanism that counters iron-driven oxidative inhibition of the liver glucose-6-phosphatase (G6Pase), and in doing so, sustains endogenous glucose production via liver gluconeogenesis. This is required to prevent the development of hypoglycemia that otherwise compromises disease tolerance to sepsis. FTH overexpression or ferritin administration establish disease tolerance therapeutically. In conclusion, disease tolerance to sepsis relies on a crosstalk between adaptive responses controlling iron and glucose metabolism, required to maintain blood glucose within a physiologic range compatible with host survival.

Microbiota's No Wasting Policy.

Schieber et al. demonstrate that a specific gut microbiota bacterial strain induces a host-mediated protection mechanism against inflammation-driven wasting syndrome. This salutary effect confers a net survival advantage against bacterial infection, without interfering with the host's pathogen load, revealing that host-microbiota interactions regulate disease tolerance to infection.

Gut microbiota elicits a protective immune response against malaria transmission.

Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:ß-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galß1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

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.

Sickle hemoglobin confers tolerance to Plasmodium infection.

Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.

Macrophages and Iron Metabolism.

Iron is a transition metal that due to its inherent ability to exchange electrons with a variety of molecules is essential to support life. In mammals, iron exists mostly in the form of heme, enclosed within an organic protoporphyrin ring and functioning primarily as a prosthetic group in proteins. Paradoxically, free iron also has the potential to become cytotoxic when electron exchange with oxygen is unrestricted and catalyzes the production of reactive oxygen species. These biological properties demand that iron metabolism is tightly regulated such that iron is available for core biological functions while preventing its cytotoxic effects. Macrophages play a central role in establishing this delicate balance. Here, we review the impact of macrophages on heme-iron metabolism and, reciprocally, how heme-iron modulates macrophage function.

Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.

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.

"Nuts and bolts" of disease tolerance.

Disease tolerance describes the ability of an infected host to limit disease severity without negatively impacting the causative pathogen. Bessede et al. (2014) show that the aryl hydrocarbon receptor is an essential component of disease tolerance during bacterial infection in mice.

Anthracyclines induce DNA damage response-mediated protection against severe sepsis.

Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.

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.

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.

Innate Nutritional Immunity.

Iron (Fe) is an essential micronutrient for both microbes and their hosts. The biologic importance of Fe derives from its inherent ability to act as a universal redox catalyst, co-opted in a variety of biochemical processes critical to maintain life. Animals evolved several mechanisms to retain and limit Fe availability to pathogenic microbes, a resistance mechanism termed "nutritional immunity." Likewise, pathogenic microbes coevolved to deploy diverse and efficient mechanisms to acquire Fe from their hosts and in doing so overcome nutritional immunity. In this review, we discuss how the innate immune system regulates Fe metabolism to withhold Fe from pathogenic microbes and how strategies used by pathogens to acquire Fe circumvent these resistance mechanisms.

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.

Tissue damage control in disease tolerance.

Immune-driven resistance mechanisms are the prevailing host defense strategy against infection. By contrast, disease tolerance mechanisms limit disease severity by preventing tissue damage or ameliorating tissue function without interfering with pathogen load. We propose here that tissue damage control underlies many of the protective effects of disease tolerance. We explore the mechanisms of cellular adaptation that underlie tissue damage control in response to infection as well as sterile inflammation, integrating both stress and damage responses. Finally, we discuss the potential impact of targeting these mechanisms in the treatment of disease.

Heme oxygenase 1 controls early innate immune response of macrophages to Salmonella Typhimurium infection.

Macrophages are central for the immune control of intracellular microbes. Heme oxygenase 1 (HO-1, hmox) is the first and rate limiting enzyme in the breakdown of heme originating from degraded senescent erythrocytes and heme-proteins, yielding equal amounts of iron, carbon monoxide and biliverdin. HO-1 is strongly up-regulated in macrophages in response to inflammatory signals, including bacterial endotoxin. In view of the essential role of iron for the growth and proliferation of intracellular bacteria along with known effects of the metal on innate immune function, we examined whether HO-1 plays a role in the control of infection with the intracellular bacterium Salmonella Typhimurium. We studied the course of infection in stably-transfected murine macrophages (RAW264.7) bearing a tetracycline-inducible plasmid producing hmox shRNA and in primary HO-1 knockout macrophages. While uptake of bacteria into macrophages was not affected, a significantly reduced survival of intracellular Salmonella was observed upon hmox knockdown or pharmacological hmox inhibition, which was independent of Nramp1 functionality. This could be traced to limitation of iron availability for intramacrophage bacteria along with enhanced stimulation of innate immune effector pathways, including the formation of reactive oxygen and nitrogen species and increased TNF-α expression. Mechanistically, these latter effects result from intracellular iron limitation with subsequent activation of NF-κB and further inos, tnfa and p47phox transcription along with reduced formation of the anti-inflammatory and radical scavenging molecules, CO and biliverdin as a consequence of HO-1 silencing. Taken together our data provide novel evidence that the infection-driven induction of HO-1 exerts detrimental effects in the early control of Salmonella infection, whereas hmox inhibition can favourably modulate anti-bacterial immune effector pathways of macrophages and promote bacterial elimination.

The Iron age of host-microbe interactions.

Microbes exert a major impact on human health and disease by either promoting or disrupting homeostasis, in the latter instance leading to the development of infectious diseases. Such disparate outcomes are driven by the ever-evolving genetic diversity of microbes and the countervailing host responses that minimize their pathogenic impact. Host defense strategies that limit microbial pathogenicity include resistance mechanisms that exert a negative impact on microbes, and disease tolerance mechanisms that sustain host homeostasis without interfering directly with microbes. While genetically distinct, these host defense strategies are functionally integrated, via mechanisms that remain incompletely defined. Here, we explore the general principles via which host adaptive responses regulating iron (Fe) metabolism impact on resistance and disease tolerance to infection.

Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity.

Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (Δhmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in Δhmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4(+) and CD8(+) T cells, including CD8(+) granzyme B(+) and CD8(+) IFN-γ(+) cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.