Celine S. Lages

Functional Regulatory T Cells Accumulate in Aged Hosts and Promote Chronic Infectious Disease Reactivation

Declines in immune function are well described in the elderly and are considered to contribute significantly to the disease burden in this population. Regulatory T cells (Tregs), a CD4+ T cell subset usually characterized by high CD25 expression, control the intensity of immune responses both in rodents and humans. However, because CD25 expression does not define all Tregs, especially in aged hosts, we characterized Tregs by the expression of FOXP3, a transcription factor crucial for Treg differentiation and function. The proportion of FOXP3+CD4+ Tregs increased in the blood of the elderly and the lymphoid tissues of aged mice. The expression of functional markers, such as CTLA-4 and GITR, was either preserved or increased on FOXP3+ Tregs from aged hosts, depending on the tissue analyzed. In vitro depletion of peripheral Tregs from elderly humans improves effector T cell responses in most subjects. Importantly, Tregs from old FoxP3-GFP knock-in mice were suppressive, exhibiting a higher level of suppression per cell than young Tregs. The increased proportion of Tregs in aged mice was associated with the spontaneous reactivation of chronic Leishmania major infection in old mice, likely because old Tregs efficiently suppressed the production of IFN-γ by effector T cells. Finally, in vivo depletion of Tregs in old mice attenuated disease severity. Accumulation of functional Tregs in aged hosts could therefore play an important role in the frequent reactivation of chronic infections that occurs in aging. Manipulation of Treg numbers and/or activity may be envisioned to enhance the control of infectious diseases in this fragile population.

A Major Role for Bim in Regulatory T Cell Homeostasis

We have previously shown that regulatory T cells (Treg) accumulate dramatically in aged animals and negatively impact the ability to control persistent infection. However, the mechanisms underlying the age-dependent accrual of Treg remain unclear. In this study, we show that Treg accumulation with age is progressive and likely not the result of increased thymic output, increased peripheral proliferation, or from enhanced peripheral conversion. Instead, we found that Treg from aged mice are more resistant to apoptosis than Treg from young mice. Although Treg from aged mice had increased expression of functional IL-7Rα, we found that IL-7R signaling was not required for maintenance of Treg in vivo. Notably, aged Treg exhibit decreased expression of the proapoptotic molecule Bim compared with Treg from young mice. Furthermore, in the absence of Bim, Treg accumulate rapidly, accounting for >25% of the CD4+ T cell compartment by 6 mo of age. Additionally, accumulation of Treg in Bim-deficient mice occurred after the cells left the transitional recent thymic emigrant compartment. Mechanistically, we show that IL-2 drives preferential proliferation and accumulation of Bimlo Treg. Collectively, our data suggest that chronic stimulation by IL-2 leads to preferential expansion of Treg having low expression of Bim, which favors their survival and accumulation in aged hosts.

Pharmacological inhibition of apical sodium‐dependent bile acid transporter changes bile composition and blocks progression of sclerosing cholangitis in multidrug resistance 2 knockout mice

Deficiency of multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leads to excretion of low‐phospholipid “toxic” bile causing progressive cholestasis. We hypothesize that pharmacological inhibition of the ileal, apical sodium‐dependent bile acid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2–/– mice. Thirty‐day‐old, female mdr2–/– mice were fed high‐fat chow containing 0.006% SC‐435, a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/day of compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry.

Partial restoration of T cell function in aged mice by in vitro blockade of the PD-1/PD-L1 pathway

Programmed cell death‐1 (PD‐1) is a newly characterized negative regulator of immune responses. The interaction of PD‐1 with its ligands (PD‐L1 and PD‐L2) inhibits T‐cell proliferation and cytokine production in young mice. Increased PD‐1 expression has been described during chronic infections, inducing chronic activation of the immune system to control it. As aging is associated with chronic immune activation, PD‐1 may contribute to age‐associated T‐cell dysfunction. Our data showed the following results in aged mice: (i) the number of PD‐1‐expressing T cells and the level of expression of PD‐Ls was increased on dendritic cell subsets and T cells; (ii) PD‐1+ T cells were exhausted effector memory T cells, as shown by their lower level of CD127, CD25 and CD28, as well as their limited proliferative and cytokine‐producing capacity; (iii) the expression of PD‐1 was up‐regulated after T‐cell receptor‐mediated activation of CD8+ T cells, but not of CD4+ T cells; (iv) blockade of the PD‐1/PD‐L1 pathway moderately improved the cytokine production of T cells from old mice but did not restore their proliferation; and (v) blockade of the PD‐1/PD‐L1 pathway did not restore function of PD‐1+ T cells; its effect appeared to be exclusively mediated by increased functionality of the PD‐1− T cells. Our data thus suggest that blockade of the PD‐1/PD‐L1 is not likely to be efficient at restoring exhausted T‐cell responses in aged hosts, although improving the responses of PD‐1− T cells may prove to be a helpful strategy in enhancing primary responses.

Regulatory T cells control the CD8 adaptive immune response at the time of ductal obstruction in experimental biliary atresia.

CD8 T‐lymphocytes are effector cells of cholangiocyte injury in human and in rhesus rotavirus (RRV)‐induced experimental biliary atresia (BA). Here we hypothesize that neonatal deficiency in CD25+CD4+ regulatory T cells (Tregs) leads to aberrant activation of hepatic T‐lymphocytes in BA. We found that adoptive transfer of total CD4 cells, but not of CD25‐depleted CD4 cells, prior to RRV inoculation reduced expansion of CD8 cells, plasma bilirubin levels, ductal inflammation, and bile duct epithelial injury at 7 days postinfection (dpi) compared with age‐matched infected controls without adoptive transfer. Searching for mechanisms, we found that in vitro production of interferon‐gamma (IFN‐γ) by naïve CD8 cells upon polyclonal stimulation was enhanced in coculture with hepatic dendritic cells (DCs) from RRV‐infected, but not with DCs from noninfected mice, which was correlated with an increased proportion of CD11b+ myeloid (m)DCs and up‐regulation of the costimulatory molecule CD86 on RRV‐primed DCs. Furthermore, DC‐dependent T‐lymphocyte activation was blocked by anti‐CD86 antibody in dose‐dependent fashion. Importantly, expression of CD86 on mDCs was down‐regulated by Tregs in vitro, and adoptive transfer of Treg‐containing CD4 cells decreased expression of CD86 on hepatic mDCs at 7 dpi. On the contrary, in mice resistant to experimental BA, CD25+ cell depletion aggravated bile duct injury at 12 dpi after RRV inoculation, as plasma bilirubin levels were elevated by >20‐fold compared with nondepleted infected controls. Increased susceptibility to hepatobiliary injury in Treg‐depleted mice was linked to hepatic CD8 expansion and enhanced stimulatory capacity of hepatic DCs. Conclusion: Activation of hepatic T‐lymphocytes driving biliary obstruction in BA is regulated by mDCs by way of CD86‐dependent costimulation and is susceptible to inhibition by Tregs. (HEPATOLOGY 2012;56:219–227)

Loss of Phagocytic and Antigen Cross-Presenting Capacity in Aging Dendritic Cells Is Associated with Mitochondrial Dysfunction.

Impaired functionality of dendritic cells (DCs) significantly contributes to decreased adaptive immune responses in aged hosts. The expression of MHC-peptide on the DC surface is the critical first step in T cell priming, but few studies have addressed the effect of aging on Ag acquisition, processing, and presentation by DCs. In this study, we show that aged murine DCs were less efficient in the cross-presentation of cell-associated Ag and subsequently in the cross-priming of CD8+ T cells than were their young counterparts. The decreased cross-presentation was associated with a reduction in the frequency of CD8α DCs and merocytic (CD8α−CD11b−)DCs that could endocytose cell-associated Ag, as well as the number and the size of the endocytosed particles in the DC that did internalize cell-associated materials. Mechanistically, phagocytic capacity has been associated with mitochondrial activity and membrane potential (Δψm). Aged DCs exhibited profound signs of mitochondrial dysfunction, illustrated by lower Δψm, reduced ATP turnover and coupling efficiency, decreased baseline oxidative phosphorylation, and greater proton leak and reactive oxygen species (ROS) production. Mimicking the aged metabolic phenotype in young DCs by pharmacologic manipulation indicated that the reductions in Δψm and ATP impeded the phagocytic capacity whereas ROS interfered with a later step in the cross-presentation process. Conversely, in vitro scavenging of ROS partially restored cross-presentation by aged DCs. Taken together, these data suggest that improvement of aged DC functionality might be feasible in the elderly by targeting metabolic dysfunction or its downstream sequelae, thereby opening new avenues for enhancing vaccine efficiency in this population.

The dendritic cell–T helper 17–macrophage axis controls cholangiocyte injury and disease progression in murine and human biliary atresia

Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL‐17A)‐green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL‐17 receptor A−/− mice were used to examine T‐lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus–induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4+ lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL‐17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c+ dendritic cells diminished hepatic IL‐17A production and ameliorated intrahepatic bile duct injury. Recombinant IL‐17A induced expression of chemokine (C‐C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C‐C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL‐17A signaling was associated with down‐regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6Chi macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL‐17 receptor A, and the prevalence of IL‐17A+ cells was positively correlated with the degree of CD68+ macrophage infiltration at diagnosis. Hepatic CD4+ lymphocytes were chief producers of IL‐17A in patients with progressive disease undergoing liver transplantation. Conclusion: These findings identify the dendritic cell–T helper 17–macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174‐188).

RHESUS ROTAVIRUS VP4 SEQUENCE-SPECIFIC ACTIVATION OF MONONUCLEAR CELLS IS ASSOCIATED WITH CHOLANGIOPATHY IN MURINE BILIARY ATRESIA

Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRVs VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

Hepatic MDR3 expression impacts lipid homeostasis and susceptibility to inflammatory bile duct obstruction in neonates

BackgroundHeterozygous mutations in the gene ABCB4, encoding the phospholipid floppase MDR3 (Mdr2 in mice), are associated with various chronic liver diseases. Here we hypothesize that reduced ABCB4 expression predisposes to extrahepatic biliary atresia (EHBA).MethodsLivers from neonatal wild-type (wt) and heterozygous Mdr2-deficient mice were subjected to mass spectrometry-based lipidomics and RNA sequencing studies. Following postnatal infection with rhesus rotavirus (RRV), liver immune responses and EHBA phenotype were assessed. Hepatic microarray data from 40 infants with EHBA were mined for expression levels of ABCB4.ResultsPhosphatidylcholine (PC) and phosphatidylethanolamine (PE) were increased, whereas the PC/PE ratio was decreased in neonatal Mdr2+/− mice compared with wt mice. Following RRV challenge, hepatic expression of IFNγ and infiltration with CD8+ and NK+ lymphocytes were increased in Mdr2+/− mice. Plasma total bilirubin levels and prevalence of complete ductal obstruction were higher in these mice. In infants with EHBA, hepatic gene expression of ABCB4 was downregulated in those with an inflammatory compared with a fibrosing molecular phenotype.ConclusionDecreased expression of ABCB4 causes dysregulation in (phospho)lipid homeostasis, and predisposes to aberrant pro-inflammatory lymphocyte responses and an aggravated phenotype of EHBA in neonatal mice. Downregulated ABCB4 is associated with an inflammatory transcriptome signature in infants with EHBA.

Interleukin 2 promotes hepatic regulatory T cell responses and protects from biliary fibrosis in murine sclerosing cholangitis.

In the multidrug resistance protein 2 (Mdr2)‐/‐ mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2‐/‐ mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14‐30 of life. Antibody‐mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro‐fibrogenic cytokine. Depletion of intrahepatic Tregs with anti‐CD25 antibody between days 7‐30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti‐interleukin 2 immune complexes (IL‐2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL‐2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro‐inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2‐/‐ mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg‐associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL‐2c, suggesting that Treg‐directed low‐dose Il‐2 treatment may be considered as therapy for SC.