Joseph Barbi

Control of TH17/Treg Balance by Hypoxia-Inducible Factor 1

T cell differentiation into distinct functional effector and inhibitory subsets is regulated, in part, by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between regulatory T cell (T(reg)) and T(H)17 differentiation. HIF-1 enhances T(H)17 development through direct transcriptional activation of RORγt and via tertiary complex formation with RORγt and p300 recruitment to the IL-17 promoter, thereby regulating T(H)17 signature genes. Concurrently, HIF-1 attenuates T(reg) development by binding Foxp3 and targeting it for proteasomal degradation. Importantly, this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α-deficient T cells are resistant to induction of T(H)17-dependent experimental autoimmune encephalitis associated with diminished T(H)17 and increased T(reg) cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.

Glucose-Independent Glutamine Metabolism via TCA Cycling for Proliferation and Survival in B Cells

Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using stable isotope-resolved metabolomics. Using [U-(13)C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-(13)C,(15)N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their (13)C-labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency makes them susceptible to the glutaminase inhibitor BPTES and hence could be targeted for cancer therapy.

Eos Mediates Foxp3-Dependent Gene Silencing in CD4+ Regulatory T Cells

Treg Responses to Eos CD4+ regulatory T cells (Tregs) are critical for keeping our immune system in check: They prevent immune responses from getting out of hand and keep autoimmunity at bay. By activating the expression of some genes and turning off expression of others, the master regulatory transcription factor of Tregs, Foxp3, endows these cells with the appropriate gene expression program to mediate their suppressive effects. Pan et al. (p. 1142, published online 20 August) now demonstrate that the transcription factor Eos is selectively required for Foxp3-mediated gene suppression in mice. Genes normally suppressed by Foxp3 in Tregs remained “on” when Eos expression was suppressed, whereas genes activated by Foxp3 were unaffected. Treg function was also affected by Eos suppression. With half their genetic program disrupted, these cells resembled an intermediate between Tregs and conventional CD4+ T cells—unable to suppress immune responses properly and partially responsive to T cell–activating stimulation. A transcription factor required for gene suppression in regulatory T cells is identified. CD4+ regulatory T cells (Tregs) maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. The core genetic program of Tregs and their ability to suppress pathologic immune responses depends on the transcription factor Foxp3. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. We identified Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Tregs. Eos interacts directly with Foxp3 and induces chromatin modifications that result in gene silencing in Tregs. Silencing of Eos in Tregs abrogates their ability to suppress immune responses and endows them with partial effector function, thus demonstrating the critical role that Eos plays in Treg programming.

Metabolic control of the Treg/Th17 axis

The interplay of the immune system with other aspects of physiology is continually being revealed and in some cases studied in considerable mechanistic detail. A prime example is the influence of metabolic cues on immune responses. It is well appreciated that upon activation, T cells take on a metabolic profile profoundly distinct from that of their quiescent and anergic counterparts; however, a number of recent breakthroughs have greatly expanded our knowledge of how aspects of cellular metabolism can shape a T‐cell response. Particularly important are findings that certain environmental cues can tilt the delicate balance between inflammation and immune tolerance by skewing T‐cell fate decisions toward either the T‐helper 17 (Th17) or T‐regulatory (Treg) cell lineage. Recognizing the unappreciated immune‐modifying potential of metabolic factors and particularly those involved in the generation of these functionally opposing T‐cell subsets will likely add new and potent therapies to our repertoire for treating immune mediated pathologies. In this review, we summarize and discuss recent findings linking certain metabolic pathways, enzymes, and by‐products to shifts in the balance between Th17 and Treg cell populations. These advances highlight numerous opportunities for immune modulation.

Treg functional stability and its responsiveness to the microenvironment.

Regulatory T cells (Tregs) prevent autoimmunity and tissue damage resulting from excessive or unnecessary immune activation through their suppressive function. While their importance for proper immune control is undeniable, the stability of the Treg lineage has recently become a controversial topic. Many reports have shown dramatic loss of the signature Treg transcription factor Forkhead box protein 3 (Foxp3) and Treg function under various inflammatory conditions. Other recent studies demonstrate that most Tregs are extremely resilient in their expression of Foxp3 and the retention of suppressive function. While this debate is unlikely to be settled in the immediate future, improved understanding of the considerable heterogeneity within the Foxp3+ Treg population and how Treg subsets respond to ranging environmental cues may be keys to reconciliation. In this review, we discuss the diverse mechanisms responsible for the observed stability or instability of Foxp3+ Treg identity and function. These include transcriptional and epigenetic programs, transcript targeting, and posttranslational modifications that appear responsive to numerous elements of the microenvironment. These mechanisms for Treg functional modulation add to the discussion of Treg stability.

CXCR3–/– mice mount an efficient Th1 response but fail to control Leishmania major infection

Chemokines play a critical role in recruitment of leukocytes to the site of infection, which is essential for host defense. We analyzed the role of CXC chemokine receptor 3 (CXCR3) in the control of cutaneous leishmaniasis using CXCR3–/– C57BL/6 mice. We found that Leishmania major‐infected CXCR3–/– mice mount an efficient Th1 response as evident by markedly increased serum levels of Th1‐associated IgG2a and significant production of IFN‐γ and IL‐12 by the draining lymph node cells, restrict systemic spread of infection, but fail to control parasite replication at the site of infection and develop chronic non‐healing lesions. Furthermore, the inability of CXCR3–/– mice to control cutaneous L. major growth was associated with fewer CD4+ and CD8+ T cells and significantly lower levels of IFN‐γ in their lesions as compared to CXCR3+/+ mice. These results demonstrate that CXCR3 plays a critical role in the host defense against cutaneous leishmaniasis caused by L. major. Furthermore, they also suggest that the susceptibility of CXCR3–/– mice to L. major is due to impaired CD4+ and CD8+ T cell trafficking and decreased production of IFN‐γ at the site of infection rather than to their inability to mount a parasite‐specific Th1 response.

Macrophage migration inhibitory factor (MIF) is critical for the host resistance against Toxoplasma gondii

Macrophage migration inhibitory factor (MIF) exerts either a protective or a deleterious role in the immune response to different pathogens. We analyzed herein the role of MIF in the host control of toxoplasmosis using MIF−/− mice backcrossed to either the BALB/c or the C57BL/6 genetic backgrounds. Both, wild‐type (WT) BALB/c and MIF−/− BALB/c mice were susceptible to infection with highly virulent RH as well as moderately virulent ME49 strains of T. gondii.MIF−/− mice, however, showed greater liver damage and more brain cysts, produced less proinflammatory cytokines, and succumbed significantly faster than WT mice. Bone marrow‐derived dendritic cells (BMDCs) from MIF−/− mice produced less interleukin‐1β, interleukin‐12, and tumor necrosis factor‐α than WT BMDCs after stimulation with soluble Toxoplasma antigen (STAg). Similar observations were made in CD11c+ low‐density cells isolated from the spleens of MIF−/− mice challenged with STAg. MIF−/−C57BL/6 mice succumbed to ME49 infection faster than their WT counterparts. C57BL/6 mice that succumbed to infection with the ME49 strain produced less MIF than resistant BALB/c mice similarly infected. Interestingly, an analysis of brains from patients with cerebral toxoplasmosis showed low levels of MIF expression. Together, these findings demonstrate that MIF plays a critical role in mediating host resistance against T. Gondii.—Flores, M., Saavedra, R., Bautista, R., Viedma, R., Tenorio, E. P., Leng, L., Sánchez, Y., Juárez, I., Satoskar, A. A., Shenoy, A. S., Terrazas, L. I., Bucala, R., Barbi J., Satoskar, A. R., Rodriguez‐Sosa, M. Macrophage migration inhibitory factor (MIF) is critical for the host resistance against Toxoplasma gondii. FASEB J. 22, 3661–3671 (2008)

Hypoxia-inducible factor 1: A link between metabolism and T cell differentiation and a potential therapeutic target

Naïve T cells activated by antigen-presenting cells (APC) can be differentiated into at least four major types of T-helper (TH) cells: TH1, TH2, TH17 and inducible regulatory T cells (iTreg) based on their unique cytokine production profiles and characteristic functions.1 TH1 produce interferon-γ (IFNγ) and are important for protective immune responses to intracellular viral, bacterial and parasitic infection. TH2 cells produce interleukin-4 (IL-4), IL-5, IL-23 and are critical for controlling extracellular parasites such as helminthes. TH17 cells are responsible for expelling extracellular bacteria and fungi through secretion of IL-17a, IL-17f and IL-22.2 These cells however are perhaps better known for their propensity to drive autoimmune responses. Tregs including naturally occurring regulatory T cells (nTreg) play important roles in the suppressive control of both innate and adaptive immunity in vivo.3,4

Cutting Edge: STAT1 and T-bet Play Distinct Roles in Determining Outcome of Visceral Leishmaniasis Caused by Leishmania donovani

T-bet and STAT1 regulate IFN-γ gene transcription in CD4+ T cells, which mediate protection against Leishmania. Here we show that T-bet and STAT1 are required for the induction of an efficient Th1 response during Leishmania donovani infection, but they play distinct roles in determining disease outcome. Both STAT1−/− and T-bet−/− mice failed to mount a Th1 response, but STAT1−/− mice were highly resistant to L. donovani and developed less immunopathology, whereas T-bet−/− mice were highly susceptible and eventually developed liver inflammation. Adoptive cell transfer studies showed that RAG2−/− recipients receiving STAT1+/+ or STAT1−/− T cells developed comparable liver pathology, but those receiving STAT1−/− T cells were significantly more susceptible to infection. These unexpected findings reveal distinct roles for T-bet and STAT1 in mediating host immunity and liver pathology during visceral leishmaniasis.

Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.