Martijn van Herwijnen | Utrecht University (original) (raw)

Papers by Martijn van Herwijnen

Chapter 1 General Introduction 9 Chapter 2 Stress proteins are used by the immune system for cogn... more Chapter 1 General Introduction 9 Chapter 2 Stress proteins are used by the immune system for cognate interactions with anti-inflammatory regulatory T cells Chapter 3 Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis Chapter 4 De novo induced regulatory T cells after Hsp70-peptide immunization are suppressive in experimental arthritis Chapter 5 A novel TCR-transgenic mouse to study Hsp70-specific CD4 + T cells Chapter 6 Discussion List of abbreviations Nederlandse Samenvatting Dankwoord Curriculum Vitae List of publications 1 General Introduction Heat shock proteins can be targets of regulatory T cells for therapeutic intervention in rheumatoid arthritis

Journal of extracellular biology, Aug 31, 2022

Breast milk is essential for facilitating the growth and development of infants and for providing... more Breast milk is essential for facilitating the growth and development of infants and for providing immune protection against viral infections in the infant's airways. Yet, regulation of inflammation by milk components may be needed to reduce immune pathology. While milk-derived extracellular vesicles (EVs) are bestowed with immunomodulatory capacities, their role in bronchial epithelial barrier function and inflammation has not yet been examined. We hypothesised that during feeding, milk is not only ingested, but aerosols containing milk EVs are inhaled and locally delivered to the infant's airways to suppress aberrant inflammation. A bronchial epithelial model of viral infection was used to explore the direct effect of milk EVs on cellular barrier function and cytokine release during stimulation with a viral dsRNA analogue (Poly I:C). We demonstrate that milk EVs improved the dsRNA-mediated decrease in ionic barrier integrity, limited tight junction reorganisation and reduced inflammatory cytokine production (IL-6, IL-8 and TNF-α). This protective response was EV-mediated, could be successfully titrated and exhibited a time-dependent response. The results indicate that if EV-containing milk aerosols are inhaled during feeding, this may lead to protection of the airway integrity from adverse inflammatory effects. K E Y WO R D S anti-inflammatory, barrier integrity, bronchial epithelium, exosomes, extracellular vesicles, milk  This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Frontiers in Immunology, Mar 9, 2016

Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study sinc... more Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigenspecific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4 + T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4 + T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4 + CD25 + Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis.

FEBS Letters, May 23, 2013

Since the initial discovery of the protective role of heat shock protein (HSP) 60 in arthritis, T... more Since the initial discovery of the protective role of heat shock protein (HSP) 60 in arthritis, T cell recognition of endogenous HSP was found to be one of the possible underlying mechanisms. Recently we have uncovered potent disease-suppressive Tregs (anti-inflammatory immunosuppressive T cells) recognizing HSP70 self-antigens, and enabling selective targeting of such Tregs to inflamed tissues. HSP70 is a major contributor to the major histocompatibility complex (MHC) Class II ligandome and we have shown that a conserved HSP70-epitope (B29) is abundantly present in murine MHC Class II. Upon transfer, B29-induced CD4+CD25+Foxp3+T cells suppressed established proteoglycan-induced arthritis (PGIA) in mice. These self-antigen specific Tregs were activated in vivo and as little as 4.000 cells sufficed to fully inhibit arthritis. Furthermore, in vivo depletion of transferred Tregs abrogated disease suppression. Given that B29 can be presented by most human MHC class II molecules and that B29 inhibited arthritis in HLA-DQ8 (human MHC) transgenic mice, we feel that therapeutic vaccination with selected HSP peptides can be an effective route for induction of anti-inflammatory Tregs as a novel intervention in chronic inflammatory diseases.

Scientific Reports, May 30, 2023

Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease bio... more Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes. Extracellular vesicles (EVs) are submicron lipid bilayer-delimited particles naturally released by cells that act as mediators of inter-cellular communication by targeting biologically active molecules to adjacent and distant cells 1. Cells in body tissues communicate by releasing EVs into proximal body fluids, such as breast milk and blood 2. Circulating EVs can originate from cells present in the body fluids, cells lining the cavities of extruded fluids or from tissue-resident cells 2 , and for this reason they can carry body fluid-specific and tissue-specific signatures. Additionally, the molecular make-up of EVs can be affected by the status of their originating cells and, as such, EVs can be enriched or depleted for specific surface proteins, resulting in specific protein biomarker profiles associated with (patho)physiological conditions 3,4. Nowadays, EV-based biomarker discovery attracts a lot of attention for monitoring disease and health status. The promise of EV-based biomarkers resides in the unique combination of different EV molecules, resulting in a "combined" biomarker that outperforms single component-based biomarkers. To identify EV-based biomarkers of disease or disturbed homeostasis, knowing the "normal" molecular reference profile of EVs in different body fluids is of utmost importance. However, the discovery of such body fluid-specific EV profiles is complicated due to the colloidal properties of body fluids, containing non-EV particles with overlapping characteristics of EVs 5,6. For example, lipoproteins in blood and casein micelles in milk, which co-isolate to various degrees with EVs 7,8 , can act as confounders in (semi)-quantitative EV analyses. Hence for comparative analyses of EVs present in different body fluids, a tailored protocol for EV isolation might overcome these problems. In immunoassays developed for EV phenotyping, the tetraspanins CD9, CD63 and CD81 are commonly used as bona fide EV-associated markers for "total" EV detection. These tetraspanins have primary functions in EV formation, cargo selection/sorting and EV release and uptake 9. Via their extracellular domains, tetraspanins associate with other tetraspanins and surface proteins thereby forming "tetraspanin webs" resulting in membrane domains with a variety of surface protein profiles 10. Importantly, in recent years, it has been reported that

PLOS ONE, Jun 24, 2015

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experim... more Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4 + CD25naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25 + cells prior to immunization, allowing the in vivo formation of Tregs from CD4 + CD25precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4 + CD25 + T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4 + CD25-T cells.

Frontiers in Nutrition, Sep 18, 2018

Mammalian milk is not only a source of nutrition for the newborn, but also contains various compo... more Mammalian milk is not only a source of nutrition for the newborn, but also contains various components that regulate further development. For instance, milk is an abundant source of microRNAs (miRNAs), which are evolutionary conserved small non-coding RNAs that are involved in post-transcriptional regulation of target mRNA. MiRNAs present in milk can occur in extracellular vesicles (EVs), which are nanosized membrane vesicles released by many cell types as a means of intercellular communication. The membrane of EVs protects enclosed miRNAs from degradation and harbors molecules that allow specific targeting to recipient cells. Although several studies have investigated the miRNA content in milk EVs from individual species, little is known about the evolutionary conserved nature of EV-associated miRNAs among different species. In this study, we profiled the miRNA content of purified EVs from human and porcine milk. These data were compared to published studies on EVs from human, cow, porcine, and panda milk to assess the overlap in the top 20 most abundant miRNAs. Interestingly, several abundant miRNAs were shared between species (e.g., let-7 family members let-7a, let-7b, let-7f, and miR-148a). Moreover, these miRNAs have been implicated in immune-related functions and regulation of cell growth and signal transduction. The conservation of these miRNA among species, not only in their sequence homology, but also in their incorporation in milk EVs of several species, suggests that they are evolutionarily selected to regulate cell function in the newborn.

Journal of extracellular vesicles, 2014

Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an impor... more Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EVand to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at (808C or 48C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system.

Journal of Colloid and Interface Science, Nov 1, 2023

bioRxiv (Cold Spring Harbor Laboratory), May 1, 2020

Molecular & Cellular Proteomics, Nov 1, 2016

Breast milk contains several macromolecular components with distinctive functions, whereby milk f... more Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. Molecular & Cellular Proteomics 15:

International Journal of Hyperthermia, Jul 17, 2013

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by excessive immune respo... more Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by excessive immune responses resulting in inflammation of the joints. Although current therapies can be successful in dampening inflammation, a long-lived state of tolerance is seldom achieved. Therefore, novel therapies are needed that restore and maintain tolerance in patients with RA. Targeting regulatory T cells (Tregs) is a successful strategy to achieve tolerance, as was shown in studies performed in animal models and in human clinical trials. The antigen-specificity of Tregs is crucial for their effectiveness and allows for very specific targeting of these cells. However, which antigen is suitable for autoimmune diseases such as RA, for which the autoantigens are largely unknown? Heat shock proteins (HSPs) are ubiquitously expressed and can be up-regulated during inflammation. Additionally, HSPs, or HSP-derived peptides are immunogenic and can be recognised by a variety of immune cells, including Tregs. Therefore, this review highlights the potential of HSP-specific Tregs to control inflammatory immune responses. Targeting HSP-specific Tregs in RA can be achieved via the administration of HSPs (derived peptides), thereby controlling inflammatory responses. This makes HSPs attractive candidates for therapeutic intervention in chronic autoimmune diseases, with the ultimate goal of inducing long-lasting tolerance.

bioRxiv (Cold Spring Harbor Laboratory), Oct 24, 2022

The promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of ... more The promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluidspecific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, which were used to model the potential biogenesis route of different EV subsets and their cellular origin.

Expert Opinion on Therapeutic Targets, Jul 16, 2012

Circulation, Oct 31, 2006

Background-Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized L... more Background-Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis. Methods and Results-Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor Ϫ/Ϫ mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%; PϽ0.05) and progression (45%; PϽ0.05) of atherogenesis. Tolerance to oxLDL induced a significant increase in CD4 ϩ CD25 ϩ Foxp3 ϩ cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-␤ production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis. Conclusions-OxLDL-specific T cells, present in LDL receptor Ϫ/Ϫ mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4 ϩ CD25 ϩ Foxp3 ϩ regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.

Frontiers in Bioengineering and Biotechnology, Apr 13, 2021

Journal of extracellular vesicles, 2015

Journal of extracellular biology, Oct 1, 2022

This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.