Cesar M. Rueda

Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy

Trafficking from bedside to bench Typically in translational research, a discovery in cell or molecular biology is later exploited to improve patient care. Occasionally, information flows in the opposite direction. Lo et al. found that patients with an autoimmune disorder caused by deficiency of a protein called LRBA responded dramatically to the drug abatacept (see the Perspective by Sansom). Abatacept contains a segment of a potent inhibitory immune receptor, CTLA4. Experiments prompted by this observation revealed the relationship between the two proteins: LRBA controls the intracellular trafficking and degradation of CTLA4. This information may further improve patient care, because other clinically approved drugs have the desired mechanism of action with potentially fewer side effects. Science, this issue p. 436; see also p. 377 A rare autoimmune disorder is caused by aberrant degradation of a potent inhibitory immune receptor. [Also see Perspective by Sansom] Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)–immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3+ regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

Fetal Immune Response to Chorioamnionitis

Chorioamnionitis is a frequent cause of preterm birth and is associated with an increased risk for injury responses in the lung, gastrointestinal tract, brain, and other fetal organs. Chorioamnionitis is a polymicrobial nontraditional infectious disease because the organisms causing chorioamnionitis are generally of low virulence and colonize the amniotic fluid often for extended periods, and the host (mother and the fetus) does not have typical infection-related symptoms such as fever. In this review, we discuss the effects of chorioamnionitis in experimental animal models that mimic the human disease. Our focus is on the immune changes in multiple fetal organs and the pathogenesis of chorioamnionitis-induced injury in different fetal compartments. As chorioamnionitis disproportionately affects preterm infants, we discuss the relevant developmental context for the immune system. We also provide a clinical context for the fetal responses.

Phenotypical characterization of regulatory T cells in humans and rodents.

Regulatory T cells (Tregs) constitute a fascinating subpopulation of CD4+ T cells due to their ability to limit the immune response against self and non‐self antigens. Murine models and antibodies directed against surface and intracellular molecules have allowed elucidation of the mechanisms that govern their development and function. However, these markers used to their classification lack of specificity, as they can be expressed by activated T cells. Similarly, there are slight differences between animal models, in steady state and pathological conditions, anatomical localization and strategy of analysis by flow cytometry. Here, we revised the most common markers utilized for Treg typification by flow cytometry such as CD25, forkhead box protein 3 (FoxP3) and CD127, along with our data obtained in different body compartments of humans, mice and rats. Furthermore, we revised and determined the expression of other molecules important for the phenotypical characterization of Treg cells. We draw attention to the drawbacks of those markers used in chronic states of inflammation. However, until a specific marker for the identification of Tregs is discovered, the best combination of markers will depend upon the tissue or the degree of inflammation from which Tregs derive.

Effect of chorioamnionitis on regulatory T cells in moderate/late preterm neonates

Regulatory T-cells (Treg) have a protective role for the control of immune activation and tissue damage. The effects of chorioamnionitis (chorio) on Treg in moderate/late preterm newborns are not known. We hypothesized that infants exposed to chorio would have decreased Treg frequency and/or function. We isolated mononuclear cells from adult peripheral blood and cord blood from term and moderate/late preterm infants who were classified for severity of chorio exposure. Mononuclear cells were analyzed by flow cytometry for Treg frequency and phenotype. Treg suppression of activation of conventional T-cells (Tcon) was also quantified. Treg frequencies were similar in all groups of neonates, but lower than that found in adults. Newborn Treg had a naïve phenotype, with decreased levels of CD45RO, HLA-DR, CD39 and TIGIT compared to adult Treg and chorio did not affect the phenotype. Treg from preterm newborns exposed to severe chorio had higher expression of Ki67 compared to the other groups. Treg from preterm newborns were less suppressive than Treg from adults or term, and the level of suppression was reduced with severe chorio. Relative to term, Treg frequency and phenotype were not affected by prematurity and chorio but their functionality was decreased. Lower Treg activity may contribute to inflammation in newborns that is often associated with chorioamnionitis.

Neutrophil Recruitment and Activation in Decidua with Intra-Amniotic IL-1beta in the Preterm Rhesus Macaque

ABSTRACT Chorioamnionitis, an infection/inflammation of the fetomaternal membranes, is frequently associated with preterm delivery. The mechanisms of inflammation in chorioamnionitis are poorly understood. We hypothesized that neutrophils recruited to the decidua would be the major producers of proinflammatory cytokines. We injected intra-amniotic (IA) interleukin 1beta (IL-1beta) at ∼80% gestation in rhesus macaque monkeys, Macaca mulatta, delivered the fetuses surgically 24 h or 72 h after IA injections, and investigated the role of immune cells in the chorion-amnion decidua. IA IL-1beta induced a robust infiltration of neutrophils and significant increases of proinflammatory cytokines in the chorioamnion decidua at 24 h after exposure, with a subsequent decrease at 72 h. Neutrophils in the decidua were the major source of tumor necrosis factor alpha (TNFalpha) and IL-8. Interestingly, IA IL-1beta also induced a significant increase in anti-inflammatory indoleamine 2,3-dioxygenase (IDO) expression in the decidua neutrophils. The frequency of regulatory T cells (Tregs) and FOXP3 mRNA expression in the decidua did not change after IA IL-1beta injection. Collectively, our data demonstrate that in this model of sterile chorioamnionitis, the decidua neutrophils cause the inflammation in the gestational tissues but may also act as regulators to dampen the inflammation. These results help to understand the contribution of neutrophils to the pathogenesis of chorioamnionitis-induced preterm labor.

Regulatory T-Cell-Mediated Suppression of Conventional T-Cells and Dendritic Cells by Different cAMP Intracellular Pathways

Regulatory T-cells (Tregs) mediate their suppressive action by acting directly on conventional T-cells (Tcons) or dendritic cells (DCs). One mechanism of Treg suppression is the increase of cyclic adenosine 3′,5′-monophosphate (cAMP) levels in target cells. Tregs utilize cAMP to control Tcon responses, such as proliferation and cytokine production. Tregs also exert their suppression on DCs, diminishing DC immunogenicity by downmodulating the expression of costimulatory molecules and actin polymerization at the immunological synapse. The Treg-mediated usage of cAMP occurs through two major mechanisms. The first involves the Treg-mediated influx of cAMP in target cells through gap junctions. The second is the conversion of adenosine triphosphate into adenosine by the ectonucleases CD39 and CD73 present on the surface of Tregs. Adenosine then binds to receptors on the surface of target cells, leading to increased intracellular cAMP levels in these targets. Downstream, cAMP can activate the canonical protein kinase A (PKA) pathway and the exchange protein activated by cyclic AMP (EPAC) non-canonical pathway. In this review, we discuss the most recent findings related to cAMP activation of PKA and EPAC, which are implicated in Treg homeostasis as well as the functional alterations induced by cAMP in cellular targets of Treg suppression.

Neonatal regulatory T cells have reduced capacity to suppress dendritic cell function

Regulatory T cells (Treg cells) limit contact between dendritic cells (DCs) and conventional T cells (Tcons), decreasing the formation of aggregates as well as down‐modulating the expression of co‐stimulatory molecules by DCs, thus decreasing DC immunogenicity and abrogating T‐cell activation. Despite the importance of this Treg‐cell function, the capacity of Treg cells from term and preterm neonates to suppress DCs, and the suppressive mechanisms they use, are still undefined. We found that, relative to adult Treg cells, activated Treg cells from human neonates expressed lower FOXP3 and CTLA‐4, but contained higher levels of cAMP. We developed an in vitro model in which Treg function was measured at a physiological ratio of 1 Treg for 10 Tcon and 1 monocyte‐derived DC, as Treg target. Term and preterm Treg cells failed to suppress the formation of DC‐Tcon aggregates, in contrast to naïve and memory Treg cells from adults. However, neonatal Treg cells diminished DC and Tcon activation as well as actin polymerization at the immunological synapses. In addition, CTLA‐4 and cAMP were the main suppressive molecules used by neonatal Treg. Altogether, both preterm and term neonatal Treg cells appear less functional than adult Treg cells, and this defect is consistent with the general impairment of CD4 cell function in newborns.

Lipopolysaccharide-Induced Chorioamnionitis Promotes IL-1-Dependent Inflammatory FOXP3+ CD4+ T Cells in the Fetal Rhesus Macaque.

Chorioamnionitis is associated with preterm labor and fetal inflammatory response syndrome (FIRS), causing fetal organ injury and morbidity, particularly in extremely premature infants. However, the effects of inflammation on the fetal immune system remain poorly understood, due to the difficulty of studying immune development in infants. Therefore, we used the model of intra-amniotic LPS administered at ∼80% gestation in rhesus monkeys to cause chorioamnionitis and FIRS that is similar in human pathology. Importantly, the frequency of IL-17+ and IL-22+ CD4+ T cells increased in the spleen of LPS-exposed fetuses, whereas regulatory T cell (Treg) frequency decreased. These changes persisted for at least 48 h. Notably, Th17 cytokines were predominantly expressed by FOXP3+CD4+ T cells and not by their FOXP3− counterparts. Bifunctional IL-17+FOXP3+ exhibited a phenotype of inflammatory Tregs (RORcHigh/+, HeliosLow/−, IL-2+, IFN-γ+, and IL-8+) compared with typical FOXP3+ cells. Diminished splenic Treg frequency in LPS-exposed fetuses was associated with inadequate Treg generation in the thymus. Mechanistically, the emergence of inflammatory Tregs was largely dependent on IL-1 signaling. However, blockage of IL-1R signaling did not abolish the deleterious effects of LPS on Treg frequency in the thymus or spleen. Collectively, we demonstrate that a prenatal inflammatory environment leads to inadequate Treg generation in the thymus with a switch of splenic Tregs toward an inflammatory phenotype. Both processes likely contribute to the pathogenesis of chorioamnionitis. Approaches to manipulate Treg numbers and function could thus be useful therapeutically to alleviate FIRS in preterm infants.

Type I interferons regulate susceptibility to inflammation-induced preterm birth

Preterm birth (PTB) is a leading worldwide cause of morbidity and mortality in infants. Maternal inflammation induced by microbial infection is a critical predisposing factor for PTB. However, biological processes associated with competency of pathogens, including viruses, to induce PTB or sensitize for secondary bacterial infection-driven PTB are unknown. We show that pathogen/pathogen-associated molecular pattern-driven activation of type I IFN/IFN receptor (IFNAR) was sufficient to prime for systemic and uterine proinflammatory chemokine and cytokine production and induction of PTB. Similarly, treatment with recombinant type I IFNs recapitulated such effects by exacerbating proinflammatory cytokine production and reducing the dose of secondary inflammatory challenge required for induction of PTB. Inflammatory challenge-driven induction of PTB was eliminated by defects in type I IFN, TLR, or IL-6 responsiveness, whereas the sequence of type I IFN sensing by IFNAR on hematopoietic cells was essential for regulation of proinflammatory cytokine production. Importantly, we also show that type I IFN priming effects are conserved from mice to nonhuman primates and humans, and expression of both type I IFNs and proinflammatory cytokines is upregulated in human PTB. Thus, activation of the type I IFN/IFNAR axis in pregnancy primes for inflammation-driven PTB and provides an actionable biomarker and therapeutic target for mitigating PTB risk.

IL-1β induced HIF-1α inhibits the differentiation of human FOXP3 + T cells

Differentiation of regulatory Treg (Treg) in the periphery is critical to control inflammatory processes. Although polarization of inducible Treg (iTreg) often occurs in an inflammatory environment, the effects exerted by inflammation on human iTreg differentiation have not been extensively studied. We observed that IL-1β significantly reduced the frequency of FOXP3+ T cells under iTreg-polarizing conditions. Mechanistically, we show that IL-1β activated mTORC1 and downstream upregulated hypoxia inducible factor-1 (HIF-1α) expression. Using specific inhibitors, we demonstrated that both steps were critical in the deleterious effect of IL-1β on Treg differentiation. Chemical stabilization of HIF-1α by Dimethyloxalylglycine (DMOG) also significantly impaired iTreg differentiation. Interestingly, while IL-1β-treated cells exhibited only minor changes in metabolism, DMOG treatment decreased iTreg mitochondrial respiration and increased their glycolytic capacity. In conclusion, exposure to inflammatory stimuli profoundly inhibits human Treg differentiation HIF-1α dependent, suggesting that targeting HIF-1α could be a strategy to foster iTreg differentiation in an inflammatory milieu. However, IL-1β deleterious effect does not appear to be completely driven by metabolic changes. These data thus suggest that several mechanisms contribute to the regulation of iTreg differentiation, but the timing and respective requirement for each pathway vary depending on the milieu in which iTreg differentiate.