We are what we eat: macrophages and efferocytosis.

Liebold et al. recently revealed how the identity of dying cells drives distinct changes to the macrophages which engulf and clear them, a process known as efferocytosis. During infection with the helminth Schistosoma mansoni, liver macrophages recapitulate these phenotypes, mediated by Axl/MerTK receptors and regulating egg burdens.

Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-ß mimic.

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-ß mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-ß receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type-specific potency of TGF-ß signaling in mammalian cells.

Modulation of haematopoiesis by protozoal and helminth parasites.

During inflammation, haematopoietic stem cells (HSCs) in the bone marrow (BM) and periphery rapidly expand and preferentially differentiate into myeloid cells that mediate innate immune responses. HSCs can be directed into quiescence or differentiation by sensing alterations to the haematopoietic niche, including cytokines, chemokines, and pathogen-derived products. Most studies attempting to identify the mechanisms of haematopoiesis have focused on bacterial and viral infections. From intracellular protozoan infections to large multicellular worms, parasites are a global health burden and represent major immunological challenges that remain poorly defined in the context of haematopoiesis. Immune responses to parasites vary drastically, and parasites have developed sophisticated immunomodulatory mechanisms that allow development of chronic infections. Recent advances in imaging, genomic sequencing, and mouse models have shed new light on how parasites induce unique forms of emergency haematopoiesis. In addition, parasites can modify the haematopoiesis in the BM and periphery to improve their survival in the host. Parasites can also induce long-lasting modifications to HSCs, altering future immune responses to infection, inflammation or transplantation, a term sometimes referred to as central trained immunity. In this review, we highlight the current understanding of parasite-induced haematopoiesis and how parasites target this process to promote chronic infections.

TGM6, a helminth secretory product, mimics TGF-ß binding to TßRII to antagonize TGF-ß signaling in fibroblasts.

The murine helminth parasite Heligmosomoides polygyrus expresses a family of proteins structurally related to TGF-ß Mimic 1 (TGM1), a secreted five domain protein that activates the TGF-ß pathway and converts naïve T lymphocytes to immunosuppressive Tregs. TGM1 signals through the TGF-ß type I and type II receptors, TßRI and TßRII, with domains 1-2 and 3 binding TßRI and TßRII, respectively, and domains 4-5 binding CD44, a co-receptor abundant on T cells. TGM6 is a homologue of TGM1 that is co-expressed with TGM1, but lacks domains 1 and 2. Herein, we show that TGM6 binds TßRII through domain 3, but does not bind TßRI, or other type I or type II receptors of the TGF-ß family. In TGF-ß reporter assays in fibroblasts, TGM6, but not truncated TGM6 lacking domains 4 and 5, potently inhibits TGF-ß- and TGM1-induced signaling, consistent with its ability to bind TßRII but not TßRI or other receptors of the TGF-ß family. However, TGM6 does not bind CD44 and is unable to inhibit TGF-ß and TGM1 signaling in T cells. To understand how TGM6 binds TßRII, the X-ray crystal structure of the TGM6 domain 3 bound to TßRII was determined at 1.4 Å. This showed that TGM6 domain 3 binds TßRII through an interface remarkably similar to the TGF-ß:TßRII interface. These results suggest that TGM6 has adapted its domain structure and sequence to mimic TGF-ß binding to TßRII and function as a potent TGF-ß and TGM1 antagonist in fibroblasts. The coexpression of TGM6, along with the immunosuppressive TGMs that activate the TGF-ß pathway, may prevent tissue damage caused by the parasite as it progresses through its life cycle from the intestinal lumen to submucosal tissues and back again.

Trained Innate Immunity in Hematopoietic Stem Cell and Solid Organ Transplantation.

Although significant progress has been made to improve short-term survival of transplant patients, long-term acceptance of allografts in solid organ and hematopoietic stem cell (HSC) transplantation is still a significant challenge. Current therapeutics for preventing or treating allograft rejection rely on potent immunosuppressive drugs that primarily target T cells of the adaptive immune response. Promising advances in transplant immunology have highlighted the importance of innate immune responses in allograft acceptance and rejection. Recent studies have demonstrated that innate immune cells are capable of mediating memory-like responses during inflammation, a term known as trained innate immunity. In this process, innate immune cells, such as macrophages and monocytes, undergo metabolic and epigenetic changes in response to a primary stimulus with a pathogen or their products that result in faster and more robust responses to a secondary stimulus. There is also some evidence to suggest that innate immune cells or their progenitors may be more anti-inflammatory after initial stimulation with appropriate agents, such as helminth products. Although this phenomenon has primarily been studied in the context of infection, there is emerging evidence to suggest that it could play a vital role in transplantation rejection and tolerance. Mechanisms of training innate immune cells and their progenitors in the bone marrow are therefore attractive targets for mediating long-term solid organ and HSC transplant tolerance. In this review, we highlight the potential role of proinflammatory and anti-inflammatory mechanisms of trained innate immunity in solid organ and HSC transplantation.

Helminth Imprinting of Hematopoietic Stem Cells Sustains Anti-Inflammatory Trained Innate Immunity That Attenuates Autoimmune Disease.

Certain proinflammatory stimuli can metabolically and epigenetically modify monocytes/macrophages or NK cells to be more responsive to secondary stimuli, a process known as trained innate immunity. However, the longevity of trained innate immunity is unclear. In this study, we report that Fasciola hepatica excretory-secretory products (FHES) can imprint an anti-inflammatory phenotype on long-term hematopoietic stem cells (HSCs) and monocyte precursor populations, enhancing their proliferation and differentiation into anti-inflammatory Ly6Clow monocytes. These monocytes expand and populate multiple compartments within mice, conferring hyporesponsiveness to proinflammatory stimuli and reduced susceptibility to induction of experimental autoimmune encephalomyelitis. Mice treated with FHES had enhanced alternatively activated macrophages, reduced Th1 and Th17 responses, and attenuating effects on autoimmunity that persisted for 8 mo. Furthermore, transplantation of HSCs from FHES-treated mice transferred the anti-inflammatory phenotype to naive mice. Our findings demonstrate that helminth products can modulate HSCs to promote development of anti-inflammatory myeloid cells that attenuate T cell-mediated autoimmune disease.

IL-33-Stimulated Murine Mast Cells Polarize Alternatively Activated Macrophages, Which Suppress T Cells That Mediate Experimental Autoimmune Encephalomyelitis.

IL-33 is known to promote type 2 immune responses through ST2, a component of the IL-33R complex, expressed primarily on mast cells, Th2 cells, group 2 innate lymphoid cells and regulatory T cells, and to a lesser extent, on NK cells and Th1 cells. Consistent with previous studies, we found that IL-33 polarized alternatively activated macrophages (AAMΦ) in vivo. However, in vitro stimulation of murine bone marrow-derived or peritoneal macrophages with IL-33 failed to promote arginase activity or expression of YM-1 or Retnla, markers of AAMΦ. Furthermore, macrophages have low/no basal expression of ST2. This suggested that alternative activation of macrophages may involve an IL-33-responsive third-party cell. Because mast cells have the highest expression of ST2 relative to other leukocytes, we focused on this cell type. Coculture experiments showed that IL-33-stimulated mast cells polarized AAMΦ through production of soluble factors. IL-33-stimulated mast cells produced a range of cytokines, including IL-6 and IL-13. Mast cell-derived IL-13 was required for induction of AAMΦ, whereas mast cell-derived IL-6 enhanced macrophage responsiveness to IL-13 via upregulation of the IL-4Rα receptor. Furthermore, we found that AAMΦ polarized by IL-33-stimulated mast cells could suppress proliferation and IL-17 and IFN-γ production by T cells. Finally, we show that AAMΦ polarized by IL-33-stimulated mast cells attenuated the encephalitogenic function of T cells in the experimental autoimmune encephalomyelitis model. Our findings reveal that IL-33 can promote immunosuppressive responses by polarizing AAMΦ via mast cell-derived IL-6 and IL-13.

Nano-particle mediated M2 macrophage polarization enhances bone formation and MSC osteogenesis in an IL-10 dependent manner.

Engineering a pro-regenerative immune response following scaffold implantation is integral to functional tissue regeneration. The immune response to implanted biomaterials is determined by multiple factors, including biophysical cues such as material stiffness, topography and particle size. In this study we developed an immune modulating scaffold for bone defect healing containing bone mimetic nano hydroxyapatite particles (BMnP). We first demonstrate that, in contrast to commercially available micron-sized hydroxyapatite particles, in-house generated BMnP preferentially polarize human macrophages towards an M2 phenotype, activate the transcription factor cMaf and specifically enhance production of the anti-inflammatory cytokine, IL-10. Furthermore, nano-particle treated macrophages enhance mesenchymal stem cell (MSC) osteogenesis in vitro and this occurs in an IL-10 dependent manner, demonstrating a direct pro-osteogenic role for this cytokine. BMnPs were also capable of driving pro-angiogenic responses in human macrophages and HUVECs. Characterization of immune cell subsets following incorporation of functionalized scaffolds into a rat femoral defect model revealed a similar profile, with micron-sized hydroxyapatite functionalized scaffolds eliciting pro-inflammatory responses characterized by infiltrating T cells and elevated expression of M1 macrophages markers compared to BMnP functionalized scaffolds which promoted M2 macrophage polarization, tissue vascularization and increased bone volume. Taken together these results demonstrate that nano-sized Hydroxyapatite has immunomodulatory potential and is capable of directing anti-inflammatory innate immune-mediated responses that are associated with tissue repair and regeneration.