Metal Messengers: Communication in the Bacterial World through Transition-Metal-Sensing Two-Component Systems.

Bacteria survive in highly dynamic and complex environments due, in part, to the presence of systems that allow the rapid control of gene expression in the presence of changing environmental stimuli. The crosstalk between intra- and extracellular bacterial environments is often facilitated by two-component signal transduction systems that are typically composed of a transmembrane histidine kinase and a cytosolic response regulator. Sensor histidine kinases and response regulators work in tandem with their modular domains containing highly conserved structural features to control a diverse array of genes that respond to changing environments. Bacterial two-component systems are widespread and play crucial roles in many important processes, such as motility, virulence, chemotaxis, and even transition metal homeostasis. Transition metals are essential for normal prokaryotic physiological processes, and the presence of these metal ions may also influence pathogenic virulence if their levels are appropriately controlled. To do so, bacteria use transition-metal-sensing two-component systems that bind and respond to rapid fluctuations in extracytosolic concentrations of transition metals. This perspective summarizes the structural and metal-binding features of bacterial transition-metal-sensing two-component systems and places a special emphasis on understanding how these systems are used by pathogens to establish infection in host cells and how these systems may be targeted for future therapeutic developments.

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB.

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein-protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA-NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

Non-canonical LexA proteins regulate the SOS response in the Bacteroidetes.

Lesions to DNA compromise chromosome integrity, posing a direct threat to cell survival. The bacterial SOS response is a widespread transcriptional regulatory mechanism to address DNA damage. This response is coordinated by the LexA transcriptional repressor, which controls genes involved in DNA repair, mutagenesis and cell-cycle control. To date, the SOS response has been characterized in most major bacterial groups, with the notable exception of the Bacteroidetes. No LexA homologs had been identified in this large, diverse and ecologically important phylum, suggesting that it lacked an inducible mechanism to address DNA damage. Here, we report the identification of a novel family of transcriptional repressors in the Bacteroidetes that orchestrate a canonical response to DNA damage in this phylum. These proteins belong to the S24 peptidase family, but are structurally different from LexA. Their N-terminal domain is most closely related to CI-type bacteriophage repressors, suggesting that they may have originated from phage lytic phase repressors. Given their role as SOS regulators, however, we propose to designate them as non-canonical LexA proteins. The identification of a new class of repressors orchestrating the SOS response illuminates long-standing questions regarding the origin and plasticity of this transcriptional network.

The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain.

Most pathogenic bacteria require ferrous iron (Fe2+) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly conserved prokaryotic transporter of Fe2+, but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein, and FeoB, a membrane protein that translocates Fe2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that selected FeoC proteins bind O2-sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O2 to regulate Fe2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera (Vibrio cholerae) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of VcFeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of VcFeoC reveals a truncated winged ß-sheet in which the cluster-binding domain is notably absent. Using homology modeling, we predicted the structure of VcNFeoB and used docking to identify an interaction site with VcFeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of VcFeoC and contribute to a better understanding of its role vis-à-vis FeoB.

Ins and Outs: Recent Advancements in Membrane Protein-Mediated Prokaryotic Ferrous Iron Transport.

Iron is an essential nutrient for virtually every living organism, especially pathogenic prokaryotes. Despite its importance, however, both the acquisition and the export of this element require dedicated pathways that are dependent on oxidation state. Due to its solubility and kinetic lability, reduced ferrous iron (Fe2+) is useful to bacteria for import, chaperoning, and efflux. Once imported, ferrous iron may be loaded into apo and nascent enzymes and even sequestered into storage proteins under certain conditions. However, excess labile ferrous iron can impart toxicity as it may spuriously catalyze Fenton chemistry, thereby generating reactive oxygen species and leading to cellular damage. In response, it is becoming increasingly evident that bacteria have evolved Fe2+ efflux pumps to deal with conditions of ferrous iron excess and to prevent intracellular oxidative stress. In this work, we highlight recent structural and mechanistic advancements in our understanding of prokaryotic ferrous iron import and export systems, with a focus on the connection of these essential transport systems to pathogenesis. Given the connection of these pathways to the virulence of many increasingly antibiotic resistant bacterial strains, a greater understanding of the mechanistic details of ferrous iron cycling in pathogens could illuminate new pathways for future therapeutic developments.

The FeoC [4Fe-4S] Cluster Is Redox-Active and Rapidly Oxygen-Sensitive.

The acquisition of iron is essential to establishing virulence among most pathogens. Under acidic and/or anaerobic conditions, most bacteria utilize the widely distributed ferrous iron (Fe2+) uptake (Feo) system to import metabolically-required iron. The Feo system is inadequately understood at the atomic, molecular, and mechanistic levels, but we do know it is composed of a main membrane component (FeoB) essential for iron translocation, as well as two small, cytosolic proteins (FeoA and FeoC) hypothesized to function as accessories to this process. FeoC has many hypothetical functions, including that of an iron-responsive transcriptional regulator. Here, we demonstrate for the first time that Escherichia coli FeoC (EcFeoC) binds an [Fe-S] cluster. Using electronic absorption, X-ray absorption, and electron paramagnetic resonance spectroscopies, we extensively characterize the nature of this cluster. Under strictly anaerobic conditions after chemical reconstitution, we demonstrate that EcFeoC binds a redox-active [4Fe-4S]2+/+ cluster that is rapidly oxygen-sensitive and decays to a [2Fe-2S]2+ cluster (t1/2 ≈ 20 s), similar to the [Fe-S] cluster in the fumarate and nitrate reductase (FNR) transcriptional regulator. We further show that this behavior is nearly identical to the homologous K. pneumoniae FeoC, suggesting a redox-active, oxygen-sensitive [4Fe-4S]2+ cofactor is a general phenomenon of cluster-binding FeoCs. Finally, in contrast to FNR, we show that the [4Fe-4S]2+ cluster binding to FeoC is associated with modest conformational changes of the polypeptide, but not protein dimerization. We thus posit a working hypothesis in which the cluster-binding FeoCs may function as oxygen-sensitive iron sensors that fine-tune pathogenic ferrous iron acquisition.

The crystal structure of Klebsiella pneumoniae FeoA reveals a site for protein-protein interactions.

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe2+ ) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate that KpFeoA can assume both "open" and "closed" conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a "C-shaped" clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a framework for testing FeoA-FeoB interactions, which could be exploited for future antibiotic developments.

Expression and purification of functionally active ferrous iron transporter FeoB from Klebsiella pneumoniae.

The acquisition of ferrous iron (Fe2+) is an important virulence factor utilized by several hospital-acquired (nosocomial) pathogens such as Klebsiella pneumoniae to establish infection within human hosts. Virtually all bacteria use the ferrous iron transport system (Feo) to acquire ferrous iron from their environments, which are often biological niches that stabilize Fe2+ relative to Fe3+. However, the details of this process remain poorly understood, likely owing to the few expression and purification systems capable of supplying sufficient quantities of the chief component of the Feo system, the integral membrane GTPase FeoB. This bottleneck has undoubtedly hampered efforts to understand this system in order to target it for therapeutic intervention. In this study, we describe the expression, solubilization, and purification of the Fe2+ transporter from K. pneumoniae, KpFeoB. We show that this protein may be heterologously overexpressed in Escherichia coli as the host organism. After testing several different commercially-available detergents, we have developed a solubilization and purification protocol that produces milligram quantities of KpFeoB with sufficient purity for enzymatic and biophysical analyses. Importantly, we demonstrate that KpFeoB displays robust GTP hydrolysis activity (kcatGTP of ∼10-1 s-1) in the absence of any additional stimulatory factors. Our findings suggest that K. pneumoniae may be capable of using its Feo system to drive Fe2+ import in an active manner.

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P1B-type ATPase.

The P1B-ATPases, a family of transmembrane metal transporters important for transition metal homeostasis in all organisms, are subdivided into classes based on sequence conservation and metal specificity. The multifunctional P1B-4-ATPase CzcP is part of the cobalt, zinc, and cadmium resistance system from the metal-tolerant, model organism Cupriavidus metallidurans. Previous work revealed the presence of an unusual soluble metal-binding domain (MBD) at the CzcP N-terminus, but the nature, extent, and selectivity of the transmembrane metal-binding site (MBS) of CzcP have not been resolved. Using homology modeling, we show that four wholly conserved amino acids from the transmembrane (TM) domain (Met254, Ser474, Cys476, and His807) are logical candidates for the TM MBS, which may communicate with the MBD via interactions with the first TM helix. Metal-binding analyses indicate that wild-type (WT) CzcP has three MBSs, and data on N-terminally truncated (ΔMBD) CzcP suggest the presence of a single TM MBS. Electronic absorption and electron paramagnetic resonance spectroscopic analyses of ΔMBD CzcP and variant proteins thereof provide insight into the details of Co2+ coordination by the TM MBS. These spectroscopic data, combined with in vitro functional studies of WT and variant CzcP proteins, show that the side chains of Met254, Cys476, and His807 contribute to Cd2+, Co2+, and Zn2+ binding and transport, whereas the side chain of Ser474 appears to play a minimal role. By comparison to other P1B-4-ATPases, we suggest that an evolutionarily adapted flexibility in the TM region likely afforded CzcP the ability to transport Cd2+ and Zn2+ in addition to Co2+.

A new metal binding domain involved in cadmium, cobalt and zinc transport.

The P1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. Here we report the biochemical, structural and functional characterization of a new MBD from the Cupriavidus metallidurans P1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd(2+), Co(2+) or Zn(2+) ions in distinct and unique sites and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full-length CzcP, truncated CzcP and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Taken together, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P1B-ATPases.

Ethanol Interactions With Dexmethylphenidate and dl-Methylphenidate Spheroidal Oral Drug Absorption Systems in Healthy Volunteers.

BACKGROUND/PURPOSE: Ethanol coadministered with immediate-release dl-methylphenidate (dl-MPH) or dexmethylphenidate (d-MPH) significantly increases the geomean maximum plasma concentration (Cmax) of d-MPH 22% and 15%, respectively, and elevates overall drug exposure and psychostimulant effects. We asked the question: Are these ethanol-MPH interactions based more fundamentally on (1) inhibition of postabsorption d-MPH metabolism or (2) acceleration of MPH formulation gastric dissolution by ethanol in the stomach? This was investigated using the pulsatile, distinctly biphasic, spheroidal oral drug absorption systems of dl-MPH and d-MPH. METHODS: In a randomized, 4-way crossover study, 14 healthy subjects received pulsatile dl-MPH (40 mg) or d-MPH (20 mg), with or without ethanol (0.6 g/kg), dosed 4 hours later. These 4 hours allowed the delayed-release second MPH pulse to reach a more distal region of the gut to preclude gastric biopharmaceutical influences. Plasma was analyzed using a highly sensitive chiral method. Subjective/physiological effects were recorded. FINDINGS/RESULTS: Ethanol increased the second pulse of d-MPH Cmax for dl-MPH by 35% (P < 0.01) and the partial area under the plasma concentration curve from 4 to 8 hours by 25% (P < 0.05). The respective values for enantiopure d-MPH were 27% (P = 0.001) and 20% (P < 0.01). The carboxylesterase 1-mediated transesterification metabolite ethylphenidate served as a biomarker for coexposure. Ethanol significantly potentiated stimulant responses to either formulation. IMPLICATIONS/CONCLUSIONS: These findings support drug dispositional interactions between ethanol and MPH as dominant over potential biopharmaceutical considerations. Understanding the pharmacology underlying the frequent coabuse of MPH-ethanol provides rational guidance in the selection of first-line pharmacotherapy for comorbid attention-deficit/hyperactivity disorder-alcohol use disorder.

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity.

The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein. Fe(II) DGCR8 RNA-binding heme domain (Rhed) undergoes a pH-dependent transition from 6-coordinate to 5-coordinate, due to protonation and loss of a lysine ligand; the ligand bound throughout the pH change is a histidine. Fe(II) Rhed binds CO and NO from 6- and 5-coordinate states, forming common CO and NO adducts at all pHs. Fe(II)-CO Rhed is 6-coordinate, low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO. Fe(II)-NO Rhed is 5-coordinate and pH insensitive. Fe(II)-NO also forms slowly upon reaction of Fe(III) Rhed with excess NO via a stepwise process. Heme reduction by NO is rate-limiting, and the rate would be negligible at physiological NO concentrations. Importantly, in vitro pri-miRNA processing assays show that both CO- and NO-bound DGCR8 species are inactive. Fe(II), Fe(II)-CO, and Fe(II)-NO Rhed do not bear either of the cysteine ligands found in the Fe(III) state. These data support a model in which the bis-cysteine thiolate ligand environment of Fe(III) DGCR8 is necessary for establishing proper pri-miRNA binding and enabling processing activity.

A Small Molecule That Switches a Ubiquitin Ligase From a Processive to a Distributive Enzymatic Mechanism.

E3 ligases are genetically implicated in many human diseases, yet E3 enzyme mechanisms are not fully understood, and there is a strong need for pharmacological probes of E3s. We report the discovery that the HECT E3 Nedd4-1 is a processive enzyme and that disruption of its processivity by biochemical mutations or small molecules switches Nedd4-1 from a processive to a distributive mechanism of polyubiquitin chain synthesis. Furthermore, we discovered and structurally characterized the first covalent inhibitor of Nedd4-1, which switches Nedd4-1 from a processive to a distributive mechanism. To visualize the binding mode of the Nedd4-1 inhibitor, we used X-ray crystallography and solved the first structure of a Nedd4-1 family ligase bound to an inhibitor. Importantly, our study shows that processive Nedd4-1, but not the distributive Nedd4-1:inhibitor complex, is able to synthesize polyubiquitin chains on the substrate in the presence of the deubiquitinating enzyme USP8. Therefore, inhibition of E3 ligase processivity is a viable strategy to design E3 inhibitors. Our study provides fundamental insights into the HECT E3 mechanism and uncovers a novel class of HECT E3 inhibitors.

Ferric, not ferrous, heme activates RNA-binding protein DGCR8 for primary microRNA processing.

The RNA-binding protein DiGeorge Critical Region 8 (DGCR8) and its partner nuclease Drosha are essential for processing of microRNA (miRNA) primary transcripts (pri-miRNAs) in animals. Previous work showed that DGCR8 forms a highly stable and active complex with ferric [Fe(III)] heme using two endogenous cysteines as axial ligands. Here we report that reduction of the heme iron to the ferrous [Fe(II)] state in DGCR8 abolishes the pri-miRNA processing activity. The reduction causes a dramatic increase in the rate of heme dissociation from DGCR8, rendering the complex labile. Electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies indicate that reduction of the heme iron is accompanied by loss of the cysteines as axial ligands. ApoDGCR8 dimers, generated through reduction and removal of the heme, show low levels of activity in pri-miRNA processing in vitro. Importantly, ferric, but not ferrous, heme restores the activity of apoDGCR8 to the level of the native ferric complex. This study demonstrates binding specificity of DGCR8 for ferric heme, provides direct biochemical evidence for ferric heme serving as an activator for miRNA maturation, and suggests that an intracellular environment increasing the availability of ferric heme may enhance the efficiency of pri-miRNA processing.

Rapid development of exhaustion and down-regulation of eomesodermin limit the antitumor activity of adoptively transferred murine natural killer cells.

Natural killer (NK) cells are potent anti-viral and antitumor "first responders" endowed with natural cytotoxicity and cytokine production capabilities. To date, attempts to translate these promising biologic functions through the adoptive transfer of NK cells for the treatment of cancer have been of limited benefit. Here we trace the fate of adoptively transferred murine NK cells and make the surprising observation that NK cells traffic to tumor sites yet fail to control tumor growth or improve survival. This dysfunction is related to a rapid down-regulation of activating receptor expression and loss of important effector functions. Loss of interferon (IFN)γ production occurs early after transfer, whereas loss of cytotoxicity progresses with homeostatic proliferation and tumor exposure. The dysfunctional phenotype is accompanied by down-regulation of the transcription factors Eomesodermin and T-bet, and can be partially reversed by the forced overexpression of Eomesodermin. These results provide the first demonstration of NK-cell exhaustion and suggest that the NK-cell first-response capability is intrinsically limited. Further, novel approaches may be required to circumvent the described dysfunctional phenotype.

Diversity of the metal-transporting P1B-type ATPases.

The P1B-ATPases are integral membrane proteins that couple ATP hydrolysis to metal cation transport. Widely distributed across all domains of life, these enzymes have been previously shown to transport copper, zinc, cobalt, and other thiophilic heavy metals. Recent data suggest that these enzymes may also be involved in nickel and/or iron transport. Here we have exploited large amounts of genomic data to examine and classify the various P1B-ATPase subfamilies. Specifically, we have combined new methods of data partitioning and network visualization known as Transitivity Clustering and Protein Similarity Networks with existing biochemical data to examine properties such as length, speciation, and metal-binding motifs of the P1B-ATPase subfamily sequences. These data reveal interesting relationships among the enzyme sequences of previously established subfamilies, indicate the presence of two new subfamilies, and suggest the existence of new regulatory elements in certain subfamilies. Taken together, these findings underscore the importance of P1B-ATPases in homeostasis of nearly every biologically relevant transition metal and provide an updated framework for future studies.

Biomarkers of exocrine pancreatic injury in 2 rat acute pancreatitis models.

UNLABELLED: Consistent, sensitive biomarkers of exocrine pancreatic injury (EPIJ) in animal models and humans have historically represented a poorly met need for investigators and clinicians. EXPERIMENTAL DESIGN: Sprague-Dawley CD/International Genetic Standard system (IGS) rats were administered cerulein or cyanohydroxybutene (CHB) to induce EPIJ. Serum samples were taken at time points between 1- and 168-hr postinjection (PI), and rats were sacrificed between 24- and 168-hr PI. METHOD: We investigated a series of serum-based biomarkers including amylase, lipase, pancreas-enriched microRNAs (miRs) and inflammation biomarkers compared with concurrent hematology and pancreatic histology. RESULTS AND CONCLUSION: Microscopic EPIJ was not associated with consistent changes in hematology or inflammation biomarkers. Increased severity scores for EPIJ correlated with increased amylase and lipase values, although severity of EPIJ did not always correlate with the magnitude of enzyme increases. Microscopic EPIJ was most severe at 24 to 48 hr; increases in miR-216a (32-fold) and miR-375 (23-fold) were present at 24 hr and, along with enzymes, were normalized by 48 hr in the cerulein study. MiRs-216a and 375 were increased by ∼800- and 500-fold, respectively, at 24 hr while miR-375 remained elevated until 72 hr in the CHB study. Impact statement: Pancreas-enriched miRs hold promise as novel serum-based biomarkers for EPIJ.

Structural Determinants of Vibrio cholerae FeoB Nucleotide Promiscuity.

Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, including Vibrio cholerae (Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+ into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures of VcNFeoB in both the apo and GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150T VcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surround the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models of VcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes.

IL-17 gene ablation does not impact Treg-mediated suppression of graft-versus-host disease after bone marrow transplantation.

Regulatory T cell (Treg) immunotherapy is a promising strategy for the treatment of graft rejection responses and autoimmune disorders. Our and other laboratories have shown that the transfer of highly purified CD4(+)CD25(+)Foxp3(+) natural Treg can prevent lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation across both major and minor histocompatibility barriers. However, recent evidence suggests that the Treg suppressive phenotype can become unstable, a phenomenon that can culminate in Treg conversion into IL-17-producing cells. We hypothesized that the intense proinflammatory signals released during an ongoing alloreaction might redirect a fraction of the transferred Treg to the Th17 cell fate, thereby losing immunosuppressive potential. We therefore sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model. We show that although Il17 gene ablation results in a mildly enhanced Treg immunosuppressive ability in vitro, such improvement is not observed when IL-17-deficient Treg are used for GVHD suppression in vivo. Similarly, when we selectively blocked IL-1 signaling in Treg, that was shown to be necessary for Th17 conversion, we did not detect any improvement on Treg-mediated GVHD suppressive ability in vivo. Furthermore, upon ex vivo reisolation of transferred wild-type Treg, we detected little or no Treg-mediated IL-17 production upon GVHD induction. Our results indicate that blocking Th17 conversion does not affect the GVHD suppressive ability of highly purified natural Treg in vivo, suggesting that IL-17 targeting is not a valuable strategy to improve Treg immunotherapy after hematopoietic cell transplantation.

Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells.

Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4⁺CD25⁺Foxp3⁺ regulatory T-cell (Treg) proliferation and function in vitro. We investigated whether the combination of RAPA plus IL-2 could impact acute GVHD induction after bone marrow transplantation (BMT). RAPA plus IL-2 resulted in improved survival and a reduction in acute GVHD lethality associated with an increased expansion of donor type CD4⁺Foxp3⁺ Tregs and reduced CD4⁺CD25⁻ conventional T cells (Tcons). RAPA plus IL-2, but not either drug alone, increased both expansion of donor natural Tregs and conversion of induced Tregs from donor CD25⁻ Tcons while IL-2 alone increased conversion of Tregs from CD25⁻ Tcon. RAPA plus IL-2 treatment resulted in less production of IFN-γ and TNF, cytokines known to be important in the initiation of acute GVHD. These studies indicate that the pharmacologic stimulation of T cells with IL-2 and the suppression of Tcon proliferation with RAPA result in a selective expansion of functional Tregs and suppression of acute GVHD.