Annelies S. Zinkernagel

NF-κB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1α

The hypoxic response is an ancient stress response triggered by low ambient oxygen (O2) (ref. 1) and controlled by hypoxia-inducible transcription factor-1 (HIF-1), whose α subunit is rapidly degraded under normoxia but stabilized when O2-dependent prolyl hydroxylases (PHDs) that target its O2-dependent degradation domain are inhibited. Thus, the amount of HIF-1α, which controls genes involved in energy metabolism and angiogenesis, is regulated post-translationally. Another ancient stress response is the innate immune response, regulated by several transcription factors, among which NF-κB plays a central role. NF-κB activation is controlled by IκB kinases (IKK), mainly IKK-β, needed for phosphorylation-induced degradation of IκB inhibitors in response to infection and inflammation. IKK-β is modestly activated in hypoxic cell cultures when PHDs that attenuate its activation are inhibited. However, defining the relationship between NF-κB and HIF-1α has proven elusive. Using in vitro systems, it was reported that HIF-1α activates NF-κB, that NF-κB controls HIF-1α transcription and that HIF-1α activation may be concurrent with inhibition of NF-κB. Here we show, with the use of mice lacking IKK-β in different cell types, that NF-κB is a critical transcriptional activator of HIF-1α and that basal NF-κB activity is required for HIF-1α protein accumulation under hypoxia in cultured cells and in the liver and brain of hypoxic animals. IKK-β deficiency results in defective induction of HIF-1α target genes including vascular endothelial growth factor. IKK-β is also essential for HIF-1α accumulation in macrophages experiencing a bacterial infection. Hence, IKK-β is an important physiological contributor to the hypoxic response, linking it to innate immunity and inflammation.

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.

Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs)

Iron is essential for many biological processes, including oxygen delivery, and its supply is tightly regulated. Hepcidin, a small peptide synthesized in the liver, is a key regulator of iron absorption and homeostasis in mammals. Hepcidin production is increased by iron overload and decreased by anemia and hypoxia; but the molecular mechanisms that govern the hepcidin response to these stimuli are not known. Here we establish that the von Hippel-Lindau/hypoxia-inducible transcription factor (VHL/HIF) pathway is an essential link between iron homeostasis and hepcidin regulation in vivo. Through coordinate downregulation of hepcidin and upregulation of erythropoietin and ferroportin, the VHL-HIF pathway mobilizes iron to support erythrocyte production.

Cutting edge: Essential role of hypoxia inducible factor-1alpha in development of lipopolysaccharide-induced sepsis.

Sepsis, the leading cause of death in intensive care units, reflects a detrimental host response to infection in which bacteria or LPS act as potent activators of immune cells, including monocytes and macrophages. In this report, we show that LPS raises the level of the transcriptional regulator hypoxia-inducible factor-1α (HIF-1α) in macrophages, increasing HIF-1α and decreasing prolyl hydroxylase mRNA production in a TLR4-dependent fashion. Using murine conditional gene targeting of HIF-1α in the myeloid lineage, we demonstrate that HIF-1α is a critical determinant of the sepsis phenotype. HIF-1α promotes the production of inflammatory cytokines, including TNF-α, IL-1, IL-4, IL-6, and IL-12, that reach harmful levels in the host during early sepsis. HIF-1α deletion in macrophages is protective against LPS-induced mortality and blocks the development of clinical markers including hypotension and hypothermia. Inhibition of HIF-1α activity may thus represent a novel therapeutic target for LPS-induced sepsis.

Statins Enhance Formation of Phagocyte Extracellular Traps

Statins are inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent clinico-epidemiologic studies correlate patients receiving statin therapy with having reduced mortality associated with severe bacterial infection. Investigating the effect of statins on the innate immune capacity of phagocytic cells against the human pathogen Staphylococcus aureus, we uncovered a beneficial effect of statins on bacterial clearance by phagocytes, although, paradoxically, both phagocytosis and oxidative burst were inhibited. Probing instead for an extracellular mechanism of killing, we found that statins boosted the production of antibacterial DNA-based extracellular traps (ETs) by human and murine neutrophils and also monocytes/macrophages. The effect of statins to induce phagocyte ETs was linked to sterol pathway inhibition. We conclude that a drug therapy taken chronically by millions alters the functional behavior of phagocytic cells, which could have ramifications for susceptibility and response to bacterial infections in these patients.

Hypoxia inducible factor (HIF) function in innate immunity and infection

The hypoxia-inducible transcription factor (HIF-1α) is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. Recently, HIF-1α has been discovered to function as a global regulator of macrophage and neutrophil inflammatory and innate immune functions, as befits these specialized phagocytic cells who must operate effectively in the hypoxic microenvironments of infected tissues. This review summarizes current knowledge of the role of HIF-1α in mammalian innate immunity, emphasizing insight gained from conditional gene targeting of the transcription factor in the myeloid cell lineage. Dynamic changes in HIF-1α expression in the course of bacterial, viral, or parasitic infections are outlined and inferences drawn regarding the consequences for pathogen and host. A better understanding of HIF-1α function may provide novel and rational approaches for boosting innate immune function in the therapy of certain complicated infectious disease conditions.

Unsafe Sex and Increased Incidence of Hepatitis C Virus Infection among HIV-Infected Men Who Have Sex with Men: The Swiss HIV Cohort Study

BACKGROUND Data on the incidence of hepatitis C virus (HCV) infection among human immunodeficiency virus (HIV)-infected persons are sparse. It is controversial whether and how frequently HCV is transmitted by unprotected sexual intercourse. METHODS We assessed the HCV seroprevalence and incidence of HCV infection in the Swiss HIV Cohort Study between 1988 and 2004. We investigated the association of HCV seroconversion with mode of HIV acquisition, sex, injection drug use (IDU), and constancy of condom use. Data on condom use or unsafe sexual behavior were prospectively collected between 2000 and 2004. RESULTS The overall seroprevalence of HCV infection was 33% among a total of 7899 eligible participants and 90% among persons reporting IDU. We observed 104 HCV seroconversions among 3327 participants during a total follow-up time of 16,305 person-years, corresponding to an incidence of 0.64 cases per 100 person-years. The incidence among participants with a history of IDU was 7.4 cases per 100 person-years, compared with 0.23 cases per 100 person-years in patients without such a history (P<.001). In men who had sex with men (MSM) without a history of IDU who reported unsafe sex, the incidence was 0.7 cases per 100 person-years, compared with 0.2 cases per 100 person-years in those not reporting unsafe sex (P=.02), corresponding to an incidence rate ratio of 3.5 (95% confidence interval, 1.2-10.0). The hazard of acquiring HCV infection was elevated among younger participants who were MSM. CONCLUSIONS HCV infection incidence in the Swiss HIV Cohort Study was mainly associated with IDU. In HIV-infected MSM, HCV infection was associated with unsafe sex.

NOD2 contributes to cutaneous defense against Staphylococcus aureus through α-toxin-dependent innate immune activation

Staphylococcus aureus is a major cause of community-acquired and nosocomial infections including the life-threatening conditions endocarditis, necrotizing pneumonia, necrotizing fasciitis, and septicemia. Toll-like receptor (TLR)-2, a membrane-bound microbial sensor, detects staphylococcal components, but macrophages lacking TLR2 or both TLR2 and TLR4 remain S. aureus responsive, suggesting that an alternative microbial recognition receptor might be involved. The cytoplasmic sensor nucleotide-binding oligomerization domain containing (NOD) 2/caspase recruitment domain (CARD) 15 detects muramyl dipeptide from bacterial peptidoglycans and mediates cytokine responses to S. aureus in vitro, but the physiological significance of these observations is not well defined. Here we show that NOD2-deficient mice exhibit a delayed but ultimately exacerbated ulcerative response and impaired bacterial clearance after s.c. infection with S. aureus. NOD2-dependent recognition of S. aureus and muramyl dipeptide is facilitated by α-toxin (α-hemolysin), a pore-forming toxin and virulence factor of the pathogen. The action of NOD2 is dependent on IL-1β-amplified production of IL-6, which promotes rapid bacterial killing by neutrophils. These results significantly broaden the physiological importance of NOD2 in innate immunity from the recognition of bacteria that primarily enter the cytoplasm to the detection of bacteria that typically reside extracellularly and demonstrate that this microbial sensor contributes to the discrimination between commensal bacteria and bacterial pathogens that elaborate pore-forming toxins.

M1 Protein Allows Group A Streptococcal Survival in Phagocyte Extracellular Traps through Cathelicidin Inhibition

M1 protein contributes to Group A Streptococcus (GAS) systemic virulence by interfering with phagocytosis and through proinflammatory activities when released from the cell surface. Here we identify a novel role of M1 protein in the stimulation of neutrophil and mast cell extracellular trap formation, yet also subsequent survival of the pathogen within these DNA-based innate defense structures. Targeted mutagenesis and heterologous expression studies demonstrate M1 protein promotes resistance to the human cathelicidin antimicrobial peptide LL-37, an important effector of bacterial killing within such phagocyte extracellular traps. Studies with purified recombinant protein fragments mapped the inhibition of cathelicidin killing to the M1 hypervariable N-terminal domain. A survey of GAS clinical isolates found that strains from patients with necrotizing fasciitis or toxic shock syndrome were significantly more likely to be resistant to cathelicidin than GAS M types not associated with invasive disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelicidin and killing within phagocyte extracellular traps contribute to the propensity of M1 GAS strains to produce invasive infections.

The IL-8 Protease SpyCEP/ScpC of Group A Streptococcus Promotes Resistance to Neutrophil Killing

Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.