Understanding Gut Feelings: Transformations in Coping With Inflammatory Bowel Disease Among Young Adults.

Past studies have revealed a dizzying array of coping techniques employed by persons living with inflammatory bowel disease (IBD). Unfortunately, research has provided little insight into when and why individuals adopt or abandon particular coping strategies. Using a retrospective narrative approach, we explored how participants made sense of changes in their approach to coping over time. Shifts in coping strategies were associated with particular illness experiences that wrought new understandings of IBD and novel identity challenges. They followed a common processual form and were marked by a movement away from techniques of purification, normalization, and banalization toward the development of a more communicative body. This was accompanied by notable shifts in identity work. Notably, participants moved from a preoccupation with maintaining continuity and sameness to permitting their extraordinary bodies to occupy a place in their public and personal identities. Implications of this process for theory and practice are discussed.

Antigen cross-presentation: proteasome location, location, location.

Our understanding of the mechanisms by which peptides from proteins present in phagosomes and endosomes are processed and presented on MHC class I molecules, in a pathway called cross-presentation, is still incomplete. One of the main questions arising from currently proposed models is how do proteins in the phagosome lumen reach the proteasome in the cytoplasm to be processed properly. In this issue of The EMBO Journal, Sengupta et al (2019) present evidence for a surprising turn of events where, in fact, the proteasome acts within the lumen of endosomes and phagosomes.

Intestinal infection triggers Parkinson's disease-like symptoms in Pink1-/- mice.

Parkinson's disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles1,2. An early-onset form of Parkinson's disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes3. PINK1 and Parkin (encoded by PRKN) are involved in the clearance of damaged mitochondria in cultured cells4, but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo5-8. It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens9, which suggests that autoimmune mechanisms participate in the aetiology of Parkinson's disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1-/- mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8+ T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with L-DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson's disease, which highlights the relevance of the gut-brain axis in the disease10.

Mitochondrial antigen presentation: a mechanism linking Parkinson's disease to autoimmunity.

Parkinson's disease (PD) is caused by the progressive loss of dopaminergic neurons and afflicts millions of people world-wide. The current treatments address only the late motor symptoms, with no cure or preventive therapeutic approaches. The contribution of dysfunctional immune mechanisms in PD has been clearly established, with an emphasis on neuroinflammation and microglial cell activation. Recent studies have widened the involvement of the immune system in this disease by clearly showing the engagement of adaptive immunity and antigen presentation processes, directly regulated by PD-related proteins, raising the question whether PD is an autoimmune disease. The contribution of autoimmune mechanisms in PD opens novel avenues for the development of preventive therapeutic approaches.

Constructing robust selves after brain injury: positive identity work among members of a female self-help group.

Despite common experiences of identity damage, decline, and deterioration, many brain injury survivors succeed in reconstructing robust identities in the wake of injury. Yet, while this accomplishment greatly benefits survivors' quality of life, little is known about how positive identity work might be facilitated or enhanced in therapeutic institutions. Drawing on data from a women's self-help group, we argue that an egalitarian, reflective, strength-focused, and gender-segregated environment can provide female ABI (acquired brain injury) survivors with a fertile scene for identity enhancement and offer unique opportunities for collective identity development. Sociolinguistic interactional analysis revealed four types of positive identity work undertaken within the group: constructing competent selves; tempering the threat of loss and impairment; resisting infantilisation and delegitimisation; and asserting a collective gender identity. This identity work was facilitated by specific programme attributes and activities and contributed to the global project of decentring disability and destigmatising impairments and losses. We call for increased attention to identity issues in brain injury rehabilitation and argue that gender-segregated programming can provide a unique space for female survivors to construct empowering individual and collective identities after injury.

Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation.

Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology.

Reframing Narratives of Aboriginal Health Inequity: Exploring Cree Elder Resilience and Well-Being in Contexts of Historical Trauma.

A large body of literature explores historical trauma or intergenerational trauma among Aboriginal communities around the globe. This literature connects contemporary forms of social suffering and health inequity to broader historical processes of colonization and the residential school systems in Canada. There are tendencies within this literature, however, to focus on individual pathology and victimization while minimizing notions of resilience or well-being. Through a social constructionist lens, this research examined how interpersonal responses to historical traumas can be intertwined with moments of and strategies for resilience. Detailed narrative interviews occurred with four Aboriginal Cree elders living in central Saskatchewan, Canada, who all experienced historical trauma to some extent. From this analysis, we argue that health research among Aboriginal populations must be sensitive to the complex individual and social realities that necessarily involve both processes of historical and contemporary traumas as well as resilience, strength, and well-being.

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

RUN and FYVE domain-containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4.

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain-containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17-positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4-treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.

Leishmania evades host immunity by inhibiting antigen cross-presentation through direct cleavage of the SNARE VAMP8.

During phagocytosis, microorganisms are taken up by immune cells into phagosomes. Through membrane-trafficking events mediated by SNARE proteins, phagosomes fuse with lysosomes, generating degradative phagolysosomes. Phagolysosomes contribute to host immunity by linking microbial killing within these organelles with antigen processing for presentation on MHC class I or II molecules to T cells. We show that the intracellular parasite Leishmania evades immune recognition by inhibiting phagolysosome biogenesis. The Leishmania cell surface metalloprotease GP63 cleaves a subset of SNAREs, including VAMP8. GP63-mediated VAMP8 inactivation or Vamp8 disruption prevents the NADPH oxidase complex from assembling on phagosomes, thus altering their pH and degradative properties. Consequently, the presentation of exogenous Leishmania antigens on MHC class I molecules, also known as cross-presentation, is inhibited, resulting in reduced T cell activation. These findings indicate that Leishmania subverts immune recognition by altering phagosome function and highlight the importance of VAMP8 in phagosome biogenesis and antigen cross-presentation.

Quantitative proteomics reveals the induction of mitophagy in tumor necrosis factor-α-activated (TNFα) macrophages.

Macrophages play an important role in innate and adaptive immunity as professional phagocytes capable of internalizing and degrading pathogens to derive antigens for presentation to T cells. They also produce pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that mediate local and systemic responses and direct the development of adaptive immunity. The present work describes the use of label-free quantitative proteomics to profile the dynamic changes of proteins from resting and TNF-α-activated mouse macrophages. These analyses revealed that TNF-α activation of macrophages led to the down-regulation of mitochondrial proteins and the differential regulation of several proteins involved in vesicle trafficking and immune response. Importantly, we found that the down-regulation of mitochondria proteins occurred through mitophagy and was specific to TNF-α, as other cytokines such as IL-1ß and IFN-γ had no effect on mitochondria degradation. Furthermore, using a novel antigen presentation system, we observed that the induction of mitophagy by TNF-α enabled the processing and presentation of mitochondrial antigens at the cell surface by MHC class I molecules. These findings highlight an unsuspected role of TNF-α in mitophagy and expanded our understanding of the mechanisms responsible for MHC presentation of self-antigens.

Inhibition of the host translation shutoff response by herpes simplex virus 1 triggers nuclear envelope-derived autophagy.

Macroautophagy is a cellular pathway that degrades intracellular pathogens and contributes to antigen presentation. Herpes simplex virus 1 (HSV-1) infection triggers both macroautophagy and an additional form of autophagy that uses the nuclear envelope as a source of membrane. The present study constitutes the first in-depth analysis of nuclear envelope-derived autophagy (NEDA). We established LC3a as a marker that allowed us to distinguish between NEDA and macroautophagy in both immunofluorescence and flow cytometry. NEDA was observed in many different cell types, indicating that it is a general response to HSV-1 infection. This autophagic pathway is known to depend on the viral protein γ34.5, which can inhibit macroautophagy via binding to beclin-1. Using mutant viruses, we were able to show that binding of beclin-1 by γ34.5 had no effect on NEDA, demonstrating that NEDA is regulated differently than macroautophagy. Instead, NEDA was triggered in response to γ34.5 binding to protein phosphatase 1α, an interaction used by the virus to prevent host cells from shutting off protein translation. NEDA was not triggered when late viral protein production was inhibited with acyclovir or hippuristanol, indicating that the accumulation of these proteins might stress infected cells. Interestingly, expression of the late viral protein gH was sufficient to rescue NEDA in the context of infection with a virus that otherwise does not support strong late viral protein expression. We argue that NEDA is a cellular stress response triggered late during HSV-1 infection and might compensate for the viral alteration of the macroautophagic response.

A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells.

The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1ß, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase-dependent manner by serum-starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity.

Proteomics analysis of herpes simplex virus type 1-infected cells reveals dynamic changes of viral protein expression, ubiquitylation, and phosphorylation.

Herpesviruses are among the most complex and widespread human viruses and cause a number of diseases ranging from cold sores to genital infections and encephalitis. While the composition of viral particles has been studied, less is known about the expression of the whole viral proteome in infected cells. Here, we analyzed the proteome of the prototypical Herpes Simplex Virus type 1 (HSV1) in infected cells by mass spectrometry. Using a high sensitivity LTQ-Orbitrap, we achieved a very high level of protein coverage and identified a total of 67 structural and nonstructural viral proteins. We also identified 90 novel phosphorylation sites and 10 novel ubiquitylation sites on different viral proteins. Ubiquitylation was observed on nine HSV1 proteins. We identified phosphorylation sites on about half of the detected viral proteins; many of the highly phosphorylated ones are known to regulate gene expression. Treatment with inhibitors of DNA replication induced changes of both viral protein abundance and modifications, highlighting the interdependence of viral proteins during the life cycle. Given the importance of expression dynamics, ubiquitylation, and phosphorylation for protein function, these findings will serve as important tools for future studies on herpesvirus biology.

Subcellular localization of iron and heme metabolism related proteins at early stages of erythrophagocytosis.

BACKGROUND: Senescent red blood cells (RBC) are recognized, phagocytosed and cleared by tissue macrophages. During this erythrophagocytosis (EP), RBC are engulfed and processed in special compartments called erythrophagosomes. We previously described that following EP, heme is rapidly degraded through the catabolic activity of heme oxygenase (HO). Extracted heme iron is then either exported or stored by macrophages. However, the cellular localization of the early steps of heme processing and iron extraction during EP remains to be clearly defined. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of our previously described cellular model of EP, using bone marrow-derived macrophages (BMDM). The subcellular localization of both inducible and constitutive isoforms of HO (HO-1 and HO-2), of the divalent metal transporters (Nramp1, Nramp2/DMT1, Fpn), and of the recently identified heme transporter HRG-1, was followed by fluorescence and electron microscopy during the earliest steps of EP. We also looked at some ER [calnexin, glucose-6-phosphatase (G6Pase) activity] and lysosomes (Lamp1) markers during EP. In both quiescent and LPS-activated BMDM, Nramp1 and Lamp1 were shown to be strong markers of the erythrophagolysosomal membrane. HRG-1 was also recruited to the erythrophagosome. Furthermore, we observed calnexin labeling and G6Pase activity at the erythrophagosomal membrane, indicating the contribution of ER in this phagocytosis model. In contrast, Nramp2/DMT1, Fpn, HO-1 and HO-2 were not detected at the membrane of erythrophagosomes. CONCLUSIONS/SIGNIFICANCE: Our study highlights the subcellular localization of various heme- and iron-related proteins during early steps of EP, thereby suggesting a model for heme catabolism occurring outside the phagosome, with heme likely being transported into the cytosol through HRG1. The precise function of Nramp1 at the phagosomal membrane in this model remains to be determined.

Proteomic characterization of phagosomal membrane microdomains during phagolysosome biogenesis and evolution.

After their formation at the cell surface, phagosomes become fully functional through a complex maturation process involving sequential interactions with various intracellular organelles. In the last decade, series of data indicated that some of the phagosome functional properties occur in specialized membrane microdomains. The molecules associated with membrane microdomains, as well as the organization of these structures during phagolysosome biogenesis are largely unknown. In this study, we combined proteomics and bioinformatics analyses to characterize the dynamic association of proteins to maturing phagosomes. Our data indicate that groups of proteins shuffle from detergent-soluble to detergent-resistant membrane microdomains during maturation, supporting a model in which the modulation of the phagosome functional properties involves an important reorganization of the phagosome proteome by the coordinated spatial segregation of proteins.

Human invariant chain isoform p35 restores thymic selection and antigen presentation in CD74-deficient mice.

The invariant chain (Ii) has pleiotropic functions and is a key factor in antigen presentation. Ii associates with major histocompatibility complex class II molecules in the endoplasmic reticulum (ER) and targets the complex in the endocytic pathway to allow antigenic peptide loading. The human Iip35 isoform includes a cytoplasmic extension containing a di-arginine motif causing ER retention. This minor isoform does not exist in mice and its function in humans has not been thoroughly investigated. We have recently generated transgenic mice expressing Iip35 and these were crossed with Ii-deficient mice to generate animals (Tgp35/mIiKO) expressing exclusively the human isoform. In these mice, we show that Iip35 is expressed in antigen presenting cells and is inducible by interferon gamma (IFN-γ). Despite the low constitutive expression of the protein and some minor differences in the Vß repertoire of Tgp35/mIiKO mice, Iip35 restored thymic selection of CD4(+) T cells and of invariant natural killer T cells. In vitro functional assays using purified primary macrophages treated with IFN-γ showed that Iip35 allows presentation of an Ii-dependent ovalbumin T-cell epitope. Altogether, our results suggest that Iip35 is functional and does not require co-expression of other isoforms for antigen presentation.

Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome.

Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ≈ 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.

The immigration experience of Iranian Baha'is in Saskatchewan: the reconstruction of their existence, faith, and religious experience.

For approximately 150 years, Baha'is in Iran have been persecuted on the basis of their religion. Limitations to aspects of their lives have compelled them to face "civic death" or migrate to other countries. This qualitative pilot study explored the experience of forced migration and how religion attenuates the disruption to the lives of Iranian Baha'is. Adaptive strategies that four participants utilised to re-establish continuity were examined. Participants who were satisfied with their lives developed a way to allow parallel cultural traditions (Iranian and Canadian) to co-exist; those who could not integrate found it difficult to maintain a balance between these traditions.

Organelle proteomics.

Proteomics has significantly contributed to improve our understanding of cell structures and functions in the last decade. The possibility to identify large sets of proteins from minute amount of material, linked with the isolation of cellular organelles using various cell fractionation methods, has provided unique insights into the molecular mechanisms governing cell functions in health and disease. The success of this approach relies on the isolation of highly enriched cell fractions enabling the separation of organelles with minimal contamination by other cellular structures.