Amir A. Sadighi Akha

Adaptation to ER Stress Is Mediated by Differential Stabilities of Pro-Survival and Pro-Apoptotic mRNAs and Proteins

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome.

Life-Span Extension in Mice by Preweaning Food Restriction and by Methionine Restriction in Middle Age

Life span can be extended in rodents by restricting food availability (caloric restriction [CR]) or by providing food low in methionine (Meth-R). Here, we show that a period of food restriction limited to the first 20 days of life, via a 50% enlargement of litter size, shows extended median and maximal life span relative to mice from normal sized litters and that a Meth-R diet initiated at 12 months of age also significantly increases longevity. Furthermore, mice exposed to a CR diet show changes in liver messenger RNA patterns, in phosphorylation of Erk, Jnk2, and p38 kinases, and in phosphorylation of mammalian target of rapamycin and its substrate 4EBP1, HE-binding protein 1 that are not observed in liver from age-matched Meth-R mice. These results introduce new protocols that can increase maximal life span and suggest that the spectrum of metabolic changes induced by low-calorie and low-methionine diets may differ in instructive ways.

Interleukin-17 Drives Pulmonary Eosinophilia following Repeated Exposure to Aspergillus fumigatus Conidia

ABSTRACT Previous research in our laboratory has demonstrated that repeated intranasal exposure to Aspergillus fumigatus conidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper TH2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the TH2 response has begun to resolve. TH17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged with A. fumigatus conidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4+ CD25+ FoxP3+ cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving TH2-type inflammation to repeated inhalation of fungal conidia.

Age-associated changes in glycosylation of CD43 and CD45 on mouse CD4 T cells

We have recently shown that treatment of T cells from aged mice with an endopeptidase specific for O‐linked glycoproteins can restore synapse formation and early activation markers to CD4 cells from aged mice. New data show that the sialidase from Clostridium perfringens, but not from Vibrio cholerae, can increase activation of CD4 cells from both old and young mice as measured by calcium signals, expression of CD25 and CD69, and secretion of IL‐2. Lectin binding assays showed alterations with age in the levels, accessibility or conformation of multiple glycoproteins on the surface of CD4 cells. While some alterations were due to the accumulation of memory cells with age, others were age sensitive and found exclusively in the naive subset or both naive and memory subsets. Furthermore, analysis of the sialic acid links α(2,3)Gal/GalNAc and α(2,6)Gal/GalNAc in immunoprecipitated CD43 and CD45 molecules confirm that age alters the glycosylation of specific proteins that regulate TCR interaction with antigen presenting cells. These data support the idea that changes in T cell surface glycosylation could play an important role in immune senescence.

T cells in aging mice: genetic, developmental, and biochemical analyses

Summary:  A combination of approaches – gene mapping, biomarker analysis, and studies of signal transduction – has helped to clarify the mechanisms of age‐related change in mouse immune status and the implications of immune aging for late‐life disease. Mapping studies have documented multiple quantitative trait loci (QTL) that influence the levels of age‐sensitive T‐cell subsets. Some of these QTL have effects that are demonstrable in young‐adult mice (8 months of age) and others demonstrable only in middle‐aged mice (18 months). Biomarker studies show that T‐cell subset levels measured at 8 or 18 months are significant predictors of lifespan for mice dying of lymphoma, fibrosarcoma, mammary adenocarcinoma, or all causes combined. Mice whose immune systems resemble that of young animals, i.e. with low levels of CD4+ and CD8+ memory T cells and relatively high levels of CD4+ T cells, tend to outlive their siblings with the opposite subset pattern. Biochemical analyses show that T cells from aged mice show defects in the activation process within a few minutes of encountering a stimulus and that the defects precede the recognition by the T‐cell receptor of agonist peptides on the antigen‐presenting cell. Defective assembly of cytoskeletal fibers and hyperglycosylation of T‐cell surface glycoproteins contribute to the immunodeficiency state, and indeed treatment with a sialylglycoprotein endopeptidase can restore full function to CD4+ T cells from aged donors in vitro.

Repeated exposure to Aspergillus fumigatus conidia results in CD4+ T cell-dependent and -independent pulmonary arterial remodeling in a mixed Th1/Th2/Th17 microenvironment that requires interleukin-4 (IL-4) and IL-10.

ABSTRACT Pulmonary arterial remodeling is a pathological process seen in a number of clinical disease states, driven by inflammatory cells and mediators in the remodeled artery microenvironment. In murine models, Th2 cell-mediated immune responses to inhaled antigens, such as purified Aspergillus allergen, have been reported to induce remodeling of pulmonary arteries. We have previously shown that repeated intranasal exposure of healthy C57BL/6 mice to viable, resting Aspergillus fumigatus conidia leads to the development of chronic pulmonary inflammation and the coevolution of Th1, Th2, and Th17 responses in the lungs. Our objective was to determine whether repeated intranasal exposure to Aspergillus conidia would induce pulmonary arterial remodeling in this mixed Th inflammatory microenvironment. Using weekly intranasal conidial challenges, mice developed robust pulmonary arterial remodeling after eight exposures (but not after two or four). The process was partially mediated by CD4+ T cells and by interleukin-4 (IL-4) production, did not require eosinophils, and was independent of gamma interferon (IFN-γ) and IL-17. Furthermore, remodeling could occur even in the presence of strong Th1 and Th17 responses. Rather than serving an anti-inflammatory function, IL-10 was required for the development of the Th2 response to A. fumigatus conidia. However, in contrast to previous studies of pulmonary arterial remodeling driven by the A. fumigatus allergen, viable conidia also stimulated pulmonary arterial remodeling in the absence of CD4+ T cells. Remodeling was completely abrogated in IL-10−/− mice, suggesting that a second, CD4+ T cell-independent, IL-10-dependent pathway was also driving pulmonary arterial remodeling in response to repeated conidial exposure.

Heightened Induction of Proapoptotic Signals in Response to Endoplasmic Reticulum Stress in Primary Fibroblasts from a Mouse Model of Longevity

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58IPK and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.

Acute infection of mice with Clostridium difficile leads to eIF2α phosphorylation and pro-survival signalling as part of the mucosal inflammatory response

The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with Clostridium difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up‐regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro‐inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti‐microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C. difficile‐infected mice expressed similar levels of CD69 and CD25. Neither tissue had up‐regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, but neither the splicing of Xbp1 nor the up‐regulation of endoplasmic reticulum chaperones, casting doubt on the full‐fledged induction of the unfolded protein response by C. difficile. They also displayed significantly higher phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), an indication of pro‐survival signalling. These data underscore the local, innate, pro‐inflammatory nature of the response to C. difficile and highlight eIF2α phosphorylation and the interleukin‐22–pSTAT3–RegIIIγ axis as two of the pathways that could be used to contain and counteract the damage inflicted on the intestinal epithelium.

Age-related Defects in Moesin/Ezrin Cytoskeletal Signals in Mouse CD4 T cells

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.

Enhancement of CD8 T-cell function through modifying surface glycoproteins in young and old mice

Previous work from our laboratory has shown that modifying cell surface glycosylation with either a Clostridium perfringens‐derived sialidase (CP‐Siase), or an O‐linked glycoprotein endopeptidase (OSGE) can enhance the function of CD4 T cells from both young and old mice at multiple levels. Here we have re‐assessed the effect of age on CD8 T‐cell function, and examined the outcome of enzymatic treatment with CP‐Siase and OSGE on its different aspects. Pre‐treatment of CD8 T cells with either CP‐Siase or OSGE led to a significant increase in anti‐CD3‐mediated Ca2+ response in both young and old mice. Pre‐treated CD8 T cells from both age groups also displayed a significant increase in activation‐induced CD69 and CD25 expression, and produced significantly higher amounts of interleukin‐2 and interferon‐γ in comparison to their untreated counterparts. Furthermore, pretreatment with either enzyme enhanced granzyme B expression in CD8 T cells, and increased their cytolytic activity in vitro. These data support the notion that glycosylated surface proteins hinder CD8 T‐cell activation and function in both young and old mice, and raise the possibility of significantly improving CD8 T cell function in older individuals through enzymatic alteration of surface glycoproteins.