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The study aims to investigate the effect of parabiosis method on endothelial dysfunction in naturally aging mice and determine the time projections for predicted improvement in the mentioned target group.The balb/c mice were separated into six groups, these being; isochronic old, heterochronic old (HP-O), isochronic young, heterochronic young, young control, and old control. After parabiosis protocol, animals were sacrificed at the third, fifth, seventh, and ninth weeks, and their thoracic aortas were isolated. The vasodilatation and vasoconstriction responses of the vessels were detected using potassium chloride and phenylephrine, acetylcholine (ACh), and sodium nitroprusside.Aging had a significant decreasing effect on maximum ACh relaxation responses (P 0.01). However, in the HP-O group, the maximum ACh relaxation response in the third week was significantly lower (P 0.05), but this effect disappeared in the ninth week. Maximum phenylephrine contraction responses were lower in the heterochronic parabiosis group (P 0.05).ACh responses increased at the end of the ninth week in the HP-O group, therefore, the parabiosis model may have an improving effect on endothelial dysfunction seen in aging.

Kidney resident macrophages (KRMs) are involved in maintaining renal homeostasis and in controlling the pathological outcome of acute kidney injury and cystic kidney disease in mice. In adult mice, KRMs maintain their population through self-renewal with little or no input from the peripheral blood. Despite recent data suggesting that a transcriptionally similar population of KRM-like cells is present across species, the idea that they are self-renewing and minimally dependent on peripheral blood input in other species has yet to be proven due to the lack of an appropriate model and cross-species expression markers. In this study, we used our recently identified cross-species KRM cell surface markers and parabiosis surgery in inbred Lewis rats to determine if rat KRMs are maintained independent of peripheral blood input, similar to their mouse counterparts. Flow cytometry analysis indicated that parabiosis surgery in the rat results in the establishment of chimerism of T/B cells, neutrophils, and monocyte-derived infiltrating macrophages in the blood, spleen, and kidney 3 wk after parabiosis surgery. Analysis of KRMs using the cell surface markers CD81 and C1q indicated that these cells have minimal chimerism and, therefore, receive little input from the peripheral blood. These data indicate that KRM properties are conserved in at least two different species. NEW & NOTEWORTHY In this report, we performed parabiosis surgery on inbred Lewis rats and showed that rat kidney resident macrophages (KRMs), identified using our novel cross-species markers, are minimally dependent on peripheral blood input. Thus, for the first time, to our knowledge, we confirm that a hallmark of mouse KRMs is also present in KRMs isolated from another species.

Gut microbiota is increasingly linked to the development of various pulmonary diseases through a gut–lung axis. However, the mechanisms by which gut commensal microbes impact trafficking and functional transition of immune cells remain largely unknown. Using integrated microbiota dysbiosis approaches, we uncover that the gut microbiota directs the migration of group 2 innate lymphoid cells (ILC2s) from the gut to the lung through a gut–lung axis. We identify Proteobacteria as a critical species in the gut microbiome to facilitate natural ILC2 migration, and increased Proteobacteria induces IL-33 production. Mechanistically, IL-33–CXCL16 signaling promotes the natural ILC2 accumulation in the lung, whereas IL-25–CCL25 signals augment inflammatory ILC2 accumulation in the intestines upon abdominal infection, parabiosis, and cecum ligation and puncture in mice. We reveal that these two types of ILC2s play critical but distinct roles in regulating inflammation, leading to balanced host defense against infection. Overall results delineate that Proteobacteria in gut microbiota modulates ILC2 directional migration to the lung for host defense via regulation of select cytokines (IL-33), suggesting novel therapeutic strategies to control infectious diseases.

Immunosuppression of unknown aetiology is a hallmark feature of glioblastoma and is characterized by decreased CD4 T-cell counts and downregulation of major histocompatibility complex class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for glioblastoma. We recapitulated the immunosuppression observed in glioblastoma patients in the C57BL/6 mouse and investigated the aetiology of low CD4 T-cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of brain cancer, including mice harbouring GL261 glioma, B16 melanoma, and in a spontaneous model of diffuse intrinsic pontine glioma. In addition to thymic involution, we determined that tumour growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC II expression on blood leucocytes, and a modest increase in bone marrow resident CD4 T cells. Using parabiosis we report that thymic involution, declines in peripheral T-cell counts, and reduced major histocompatibility complex class II expression levels were mediated through circulating blood-derived factors. Conversely, T-cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is non-steroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the immunosuppression was not unique to cancer itself, but rather occurs in response to brain injury. Non-cancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that brain cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.

Whether disc aging is influenced by factors beyond its local environment is an important unresolved question. Here we performed heterochronic parabiosis in mice to study the effects of circulating factors in young and old blood on age-associated intervertebral disc degeneration. Compared to young isochronic pairs (Y-Y), young mice paired with old mice (Y-O) showed significant increases in levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic tissue degeneration, but negligible changes in cellular senescence markers (p16INK4a, p21Cip1). Compared to old isochronic pairs (O-O), old mice paired with young mice (O-Y) exhibited a significant decrease in expression of cellular senescence markers (p16, p21, p53), but only marginal decreases in the levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic degeneration. Thus, exposing old mice to young blood circulation greatly suppressed disc cellular senescence, but only slightly decreased disc matrix imbalance and degeneration. Conversely, exposing young mice to old blood accelerated their disc matrix imbalance and tissue degeneration, with little effects on disc cellular senescence. Thus, non-cell autonomous effects of circulating factors on disc cellular senescence and matrix homeostasis are complex and suggest that disc matrix homeostasis is modulated by systemic factors and not solely through local disc cellular senescence.

The migratory patterns of virus-specific CD8 T cells during chronic viral infection are not well understood. To address this issue, we have done parabiosis experiments during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. We found that despite the high frequency of virus-specific CD8 T cells in both lymphoid and nonlymphoid tissues there was minimal migration of virus-specific CD8 T cells between the chronically infected conjoined parabiont mice. This was in contrast to parabionts between mice that had undergone an acute LCMV infection where virus-specific CD8 T cells established equilibrium demonstrating circulation of memory T cells generated after viral clearance. We have identified a population of PD-1+ TCF1+CXCR5+Tim-3- stemlike virus-specific CD8 T cells that reside in lymphoid tissues and act as resource cells for maintaining the T cell response during chronic infection. These are the cells that proliferate and give rise to the more terminally differentiated PD-1+ CXCR5-Tim-3+ CD8 T cells. Both the stemlike CD8 T cells and their terminally differentiated progeny showed minimal migration during chronic infection and the few LCMV-specific CD8 T cells that were present in circulation were the recently emerging progeny from the stemlike CD8 T cells. The PD-1+ TCF1+CXCR5+ stemlike CD8 T cells were truly resident in lymphoid tissues and did not circulate in the blood. We propose that this residency in specialized niches within lymphoid tissues is a key aspect of their biology and is essential for maintaining their quiescence and stemlike program under conditions of a chronic viral infection.

Xinning Wang,1 Qiang Zu,1 Jinshan Lu,1 Lei Zhang,1 Qiang Zhu,1 Xuefeng Sun,2 Jun Dong1 1Department of Urology, Chinese PLA General Hospital, Beijing, People’s Republic of China; 2Department of Nephrology, Chinese PLA General Hospital, Beijing, People’s Republic of ChinaCorrespondence: Xuefeng Sun; Jun Dong Email xfssun@126.com; jundong@vip.126.comIntroduction: Our previous study revealed that a young internal environment ameliorated kidney aging by virtue of an animal model of heterochronic parabiosis and a model of heterochronic renal transplantation. In this research, we used proteome to investigate the effects of donor-recipient age difference in clinical renal transplantation.Methods: This study included 10 pairs of renal transplantation donors and recipients with an age difference of greater than 20 years to their corresponding recipients/donors. All recipients have received transplantation more than 3 years ago. Renal function and the serum/urine proteomes of the donors and recipients were analyzed.Results: The renal function was similar between the young recipients and the old donors. In contrast, the renal function of the young donors was significantly superior to that of the old recipients. Furthermore, 497 and 975 proteins were identified in the serum and urine proteomes, respectively. The content of SLC3A2 in the blood was found to be related to aging, while the contents of SERPINA1 and SERPINA3 in the urine were related to immune functions after renal transplantation.Conclusion: This study demonstrated that, in the human body, a younger internal environment could ameliorate kidney aging and provided not only clinical evidence for increasing the age limit of kidney transplant donors but also new information for kidney aging research.Keywords: renal transplantation, aging, proteome, living donor transplantation

Parasitic helminths cause significant damage as they migrate through host tissues to complete their life cycle. While chronic helminth infections are characterized by a well-described Type 2 immune response, the early, tissue-invasive stages are not well understood. Here we investigate the immune pathways activated during the early stages of Heligmosomoides polygyrus bakeri (Hpb), a natural parasitic roundworm of mice. In contrast to the Type 2 immune response present at later stages of infection, a robust Type 1 immune signature including IFNg production was dominant at the time of parasite invasion and granuloma formation. This early response was associated with an accumulation of activated Natural Killer (NK) cells, with no increase of other innate lymphoid cell populations. Parabiosis and confocal microscopy studies indicated that NK cells were recruited from circulation to the small intestine, where they surrounded parasitic larvae. NK cell recruitment required IFNγ receptor signaling, but was independent of CXCR3 expression. The depletion of tissue-infiltrating NK cells altered neither worm burden nor parasite fitness, but increased vascular injury, suggesting a role for NK cells in mediating tissue protection. Together, these data identify an unexpected role for NK cells in promoting disease tolerance during the invasive stage of an enteric helminth infection.

Mucosal healing occurs through migration and proliferation of cells within injured epithelium, yet these processes may be inadequate for mucosal healing after significant injury where the mucosa is denuded. We hypothesize that extra-intestinal cells can contribute to mucosal healing after injury to the small and large intestine. We generated parabiotic pairs between wild-type and tdTomato mice, which were then subjected to radiation-induced enteritis and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. We now show that as compared with singleton mice, mice with a parabiotic partner were protected against intestinal damage as revealed by significantly reduced weight loss, reduced expression of pro-inflammatory cytokines, reduced enterocyte apoptosis, and improved crypt proliferation. Donor cells expressed CD45-, Sca-1+, c-kit+, and CXCR4+ and accumulated around the injured crypts but did not transdifferentiate into epithelia, suggesting that extra-intestinal cells play a paracrine role in the healing response, while parabiotic pairings with Rag1-/- mice showed improved healing, indicating that adaptive immune cells were dispensable for mucosal healing. Strikingly, ablation of the bone marrow of the donor parabionts removed the protective effects. These findings reveal that the recruitment of extra-intestinal, bone marrow-derived cells into the injured intestinal mucosa can promote mucosal healing, suggesting novel therapeutic approaches for severe intestinal disease.

Consumption of a high-energy Western diet triggers mild adaptive β cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of β cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of β cells, but not that of α cells, leading to enlarged β cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of β cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse β cell failure in patients with diabetes.

To test the hypothesis that iron accumulation in tissues with age is a key harmful factor for the development of aging, we established heterochronic parabiosis-pairings and investigated changes in serum iron, the expression of major iron transport proteins and iron contents, as well as telomerase reverse transcriptase (TERT), telomerase RNA component (TERC), and telomere length in the liver, kidney and heart of Y-O(O) (old pairing with young), Y-O(Y) (young pairing with old), O-O (pairings between two old) and Y-Y (pairings between two young) mice. We demonstrated that the reduced serum iron, increased iron and reduced expression of TERT and TERC in the tissues of aged mice are reversible by exposure to a younger mouse's circulation. All of these measurements in young mice are reversible by exposure to an older mouse's circulation. Correlation analysis showed that tissue iron is negatively correlated with TERT and TERC expression in the liver, kidney and heart of parabiotic mice. These findings provide new evidence for the key role of iron in aging and also imply the existence of rejuvenating factors in young serum with an anti-ageing role that act by reversing the impaired activity of iron metabolism in old mice.

Salou et al. wondered what could differentiate MAIT and NKT cells, if not for TCR specificity. Once split according to RORγt and T-bet–expressing subsets, MAIT and NKT share almost identical transcriptional programs acquired in the thymus, which result in specific tissue residency patterns., Mucosal-associated invariant T (MAIT) cells are abundant T cells with unique specificity for microbial metabolites. MAIT conservation along evolution indicates important functions, but their low frequency in mice has hampered their detailed characterization. Here, we performed the first transcriptomic analysis of murine MAIT cells. MAIT1 (RORγtneg) and MAIT17 (RORγt+) subsets were markedly distinct from mainstream T cells, but quasi-identical to NKT1 and NKT17 subsets. The expression of similar programs was further supported by strong correlations of MAIT and NKT frequencies in various organs. In both mice and humans, MAIT subsets expressed gene signatures associated with tissue residency. Accordingly, parabiosis experiments demonstrated that MAIT and NKT cells are resident in the spleen, liver, and lungs, with LFA1/ICAM1 interactions controlling MAIT1 and NKT1 retention in spleen and liver. The transcriptional program associated with tissue residency was already expressed in thymus, as confirmed by adoptive transfer experiments. Altogether, shared thymic differentiation processes generate “preset” NKT and MAIT subsets with defined effector functions, associated with specific positioning into tissues.

Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

Anatomical positioning of memory lymphocytes within barrier tissues accelerates secondary immune responses and is thought to be essential for protection at mucosal surfaces. However, it remains unclear whether resident memory in the female reproductive tract (FRT) is required for Chlamydial immunity. Here, we describe efficient generation of tissue-resident memory CD4 T cells and memory lymphocyte clusters within the FRT after vaginal infection with Chlamydia. Despite robust establishment of localized memory lymphocytes within the FRT, naïve mice surgically joined to immune mice, or mice with only circulating immunity following intranasal immunization, were fully capable of resisting Chlamydia infection via the vaginal route. Blocking the rapid mobilization of circulating memory CD4 T cells to the FRT inhibited this protective response. These data demonstrate that secondary protection in the FRT can occur in the complete absence of tissue-resident immune cells. The ability to confer robust protection to barrier tissues via circulating immune memory provides an unexpected opportunity for vaccine development against infections of the FRT.

Anatomical positioning of memory lymphocytes within barrier tissues accelerates secondary immune responses and is thought to be essential for protection at mucosal surfaces. However, it remains unclear whether resident memory in the female reproductive tract (FRT) is required for Chlamydial immunity. Here, we describe efficient generation of tissue-resident memory CD4 T cells and memory lymphocyte clusters within the FRT after vaginal infection with Chlamydia. Despite robust establishment of localized memory lymphocytes within the FRT, naive mice surgically joined to immune mice, or mice with only circulating immunity following intranasal immunization, were fully capable of resisting Chlamydia infection via the vaginal route. Blocking the rapid mobilization of circulating memory CD4 T cells to the FRT inhibited this protective response. These data demonstrate that secondary protection in the FRT can occur in the complete absence of tissue-resident immune cells. The ability to confer robust protection to barrier tissues via circulating immune memory provides an unexpected opportunity for vaccine development against infections of the FRT.

Objective The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo. Summary of background data Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo. Methods To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells. Results Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds. Conclusion Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

The decline of muscle regenerative potential with age has been attributed to a diminished responsiveness of muscle progenitor cells (MPCs). Heterochronic parabiosis has been used as a model to study the effects of aging on stem cells and their niches. These studies have demonstrated that, by exposing old mice to a young systemic environment, aged progenitor cells can be rejuvenated. One interesting idea is that pregnancy represents a unique biological model of a naturally shared circulatory system between developing and mature organisms. To test this hypothesis, we evaluated the muscle regeneration potential of pregnant mice using a cardiotoxin (CTX) injury mouse model. Our results indicate that the pregnant mice demonstrate accelerated muscle healing compared to nonpregnant control mice following muscle injury based on improved muscle histology, superior muscle regeneration, and a reduction in inflammation and necrosis. Additionally, we found that MPCs isolated from pregnant mice display a significant improvement of myogenic differentiation capacity in vitro and muscle regeneration in vivo when compared to the MPCs from nonpregnant mice. Furthermore, MPCs from nonpregnant mice display enhanced myogenic capacity when cultured in the presence of serum obtained from pregnant mice. Our proteomics data from these studies provides potential therapeutic targets to enhance the myogenic potential of progenitor cells and muscle repair.

Rationale: Unproven theories abound regarding the long-range uptake and endocrine activity of extracellular blood-borne microRNAs into tissue. In pulmonary hypertension (PH), microRNA-210 (miR-210) in pulmonary endothelial cells promotes disease, but its activity as an extracellular molecule is incompletely defined. Objective: We investigated whether chronic and endogenous endocrine delivery of extracellular miR-210 to pulmonary vascular endothelial cells promotes PH. Methods and Results: Using miR-210 replete (wild-type [WT]) and knockout mice, we tracked blood-borne miR-210 using bone marrow transplantation and parabiosis (conjoining of circulatory systems). With bone marrow transplantation, circulating miR-210 was derived predominantly from bone marrow. Via parabiosis during chronic hypoxia to induce miR-210 production and PH, miR-210 was undetectable in knockout-knockout mice pairs. However, in plasma and lung endothelium, but not smooth muscle or adventitia, miR-210 was observed in knockout mice of WT-knockout pairs. This was accompanied by downregulation of miR-210 targets ISCU (iron-sulfur assembly proteins)1/2 and COX10 (cytochrome c oxidase assembly protein-10), indicating endothelial import of functional miR-210. Via hemodynamic and histological indices, knockout-knockout pairs were protected from PH, whereas knockout mice in WT-knockout pairs developed PH. In particular, pulmonary vascular engraftment of miR-210–positive interstitial lung macrophages was observed in knockout mice of WT-knockout pairs. To address whether engrafted miR-210–positive myeloid or lymphoid cells contribute to paracrine miR-210 delivery, we studied miR-210 knockout mice parabiosed with miR-210 WT; Cx3cr1 knockout mice (deficient in myeloid recruitment) or miR-210 WT; Rag1 knockout mice (deficient in lymphocytes). In both pairs, miR-210 knockout mice still displayed miR-210 delivery and PH, thus demonstrating a pathogenic endocrine delivery of extracellular miR-210. Conclusions: Endogenous blood-borne transport of miR-210 into pulmonary vascular endothelial cells promotes PH, offering fundamental insight into the systemic physiology of microRNA activity. These results also describe a platform for RNA-mediated crosstalk in PH, providing an impetus for developing blood-based miR-210 technologies for diagnosis and therapy in this disease.

In metazoans, specific tasks are relegated to dedicated organs that are established early in development, occupy discrete locations and typically remain fixed in size. The adult immune system arises from a centralized haematopoietic niche that maintains self-renewing potential1,2, and—upon maturation—becomes distributed throughout the body to monitor environmental perturbations, regulate tissue homeostasis and mediate organism-wide defence. Here we examine how immunity is integrated within adult mouse tissues, and address issues of durability, expansibility and contributions to organ cellularity. Focusing on antiviral T cell immunity, we observed durable maintenance of resident memory T cells up to 450 days after infection. Once established, resident T cells did not require the T cell receptor for survival or retention of a poised, effector-like state. Although resident memory indefinitely dominated most mucosal organs, surgical separation of parabiotic mice revealed a tissue-resident provenance for blood-borne effector memory T cells, and circulating memory slowly made substantial contributions to tissue immunity in some organs. After serial immunizations or cohousing with pet-shop mice, we found that in most tissues, tissue pliancy (the capacity of tissues to vary their proportion of immune cells) enables the accretion of tissue-resident memory, without axiomatic erosion of pre-existing antiviral T cell immunity. Extending these findings, we demonstrate that tissue residence and organ pliancy are generalizable aspects that underlie homeostasis of innate and adaptive immunity. The immune system grows commensurate with microbial experience, reaching up to 25% of visceral organ cellularity. Regardless of the location, many populations of white blood cells adopted a tissue-residency program within nonlymphoid organs. Thus, residence—rather than renewal or recirculation—typifies nonlymphoid immune surveillance, and organs serve as pliant storage reservoirs that can accommodate continuous expansion of the cellular immune system throughout life. Although haematopoiesis restores some elements of the immune system, nonlymphoid organs sustain an accrual of durable tissue-autonomous cellular immunity that results in progressive decentralization of organismal immune homeostasis. Investigations in mice using parabiosis and cohousing experiments reveal that nonlymphoid organs serve as reservoirs of tissue-autonomous cellular immunity, leading to the decentralization of organism-level immune homeostasis.

Innate lymphoid cells (ILCs), including ILC1s, ILC2s, and ILC3s, play critical roles in regulating immunity, inflammation, and tissue homeostasis. However, limited attention is focused on the unique phenotype of ILCs in the heart tissue. In this study, we analyzed the ILC subsets in the heart by flow cytometry and found that ILC2s were the dominant population of ILCs, while a lower proportion of type 1 ILCs (including ILC1 and NK cells) and merely no ILC3s in the heart tissue of mice. Our results show that ILC2 development kinetically peaked in heart ILC2s at the age of 4 weeks after birth and later than lung ILC2s. By conducting parabiosis experiment, we show that heart ILC2s are tissue resident cells and minimally replaced by circulating cells. Notably, heart ILC2s have unique phenotypes, such as lower expression of ICOS, CD25 (IL-2Rα), and Ki-67, higher expression of Sca-1 and GATA3, and stronger ability to produce IL-4 and IL-13. In doxorubicin-induced myocardial necroptosis model of mouse heart tissue, IL-33 mRNA expression level and ILC2s were remarkably increased. In addition, IL-4 production by heart ILC2s, but not lung ILC2s, was also dramatically increased after doxorubicin treatment. Our results demonstrate that heart-resident ILC2s showed tissue-specific phenotypes and rapidly responded to heart injury. Thus, further studies are warranted to explore the potential for IL-33-elicited ILC2s response as therapeutics for attenuating heart damage.

Heterochronic parabiosis models have been utilized to demonstrate the role of blood-borne circulating factors in systemic effects of aging. In previous studies, heterochronic parabiosis has shown positive effects across multiple tissues in old mice. More recently, a study demonstrated old blood had a more profound negative effect on muscle performance and neurogenesis of young mice. In this study, we used heterochronic parabiosis to test the hypothesis that circulating factors mediate mitochondrial bioenergetic decline, a well-established biological hallmark of aging. We examined mitochondrial morphology, expression of mitochondrial complexes, and mitochondrial respiration from skeletal muscle of mice connected as heterochronic pairs, as well as young and old isochronic controls. Our results indicate that young heterochronic mice had significantly lower total mitochondrial content and on average had significantly smaller mitochondria compared to young isochronic controls. Expression of complex IV followed a similar pattern: young heterochronic mice had a trend for lower expression compared to young isochronic controls. Additionally, respirometric analyses indicate that young heterochronic mice had significantly lower complex I, complex I + II, and maximal mitochondrial respiration and a trend for lower complex II-driven respiration compared to young isochronic controls. Interestingly, we did not observe significant improvements in old heterochronic mice compared to old isochronic controls, demonstrating the profound deleterious effects of circulating factors from old mice on mitochondrial structure and function. We also found no significant differences between the young and old heterochronic mice, demonstrating that circulating factors can be a driver of age-related differences in mitochondrial structure and function.

Background Recurrent myocardial infarction (MI) is common in patients with coronary artery disease and is associated with high mortality. Long-term reprogramming of myeloid progenitors occurs in response to inflammatory stimuli and alters the organism’s response to secondary inflammatory challenges. Objectives This study examined the effect of recurrent MI on bone marrow response and cardiac inflammation. Methods The investigators developed a surgical mouse model in which 2 subsequent MIs affected different left ventricular regions in the same mouse. Recurrent MI was induced by ligating the left circumflex artery followed by the left anterior descending coronary artery branch. The study characterized the resulting ischemia by whole-heart fluorescent coronary angiography after optical organ clearing and by cardiac magnetic resonance imaging. Results A first MI-induced bone marrow “memory” via a circulating signal, reducing hematopoietic maintenance factor expression in bone marrow macrophages. This dampened the organism’s reaction to subsequent events. Despite a similar extent of injury according to troponin levels, recurrent MI caused reduced emergency hematopoiesis and less leukocytosis than a first MI. Consequently, fewer leukocytes migrated to the ischemic myocardium. The hematopoietic response to lipopolysaccharide was also mitigated after a previous MI. The increase of white blood count in 28 patients was lower after recurrent MI compared with their first MI. Conclusions The data suggested that hematopoietic and innate immune responses are shaped by a preceding MI.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT. ispartof: CELL vol:182 issue:3 pages:625-+ ispartof: location:United States status: published

Heterotopic ossification (HO) occurs secondary to trauma, causing pain and functional limitations. Identification of the cells that contribute to HO is critical to the development of therapies. Given that innate immune cells and mesenchymal stem cells are known contributors to HO, we sought to define the contribution of these populations to HO and to identify what, if any, contribution circulating populations have to HO. A shared circulation was obtained using a parabiosis model, established between an enhanced green fluorescent protein–positive/luciferase(+) donor and a same-strain nonreporter recipient mouse. The nonreporter mouse received Achilles tendon transection and dorsal burn injury to induce HO formation. Bioluminescence imaging and immunostaining were performed to define the circulatory contribution of immune and mesenchymal cell populations. Histologic analysis showed circulating cells present throughout each stage of the developing HO anlagen. Circulating cells were present at the injury site during the inflammatory phase and proliferative period, with diminished contribution in mature HO. Immunostaining demonstrated that most early circulatory cells were from the innate immune system; only a small population of mesenchymal cells were present in the HO. We demonstrate the time course of the participation of circulatory cells in trauma-induced HO and identify populations of circulating cells present in different stages of HO. These findings further elucidate the relative contribution of local and systemic cell populations to HO.

Background: Huntington's disease (HD) starts its pathology long before clinical manifestation, however, there is no therapy to cure it completely and only a few studies have been reported for delaying the progression of HD. Recently, it has been shown that heterochronic parabiosis can modulate the neurodegenerative diseases. Despite the importance of the transportation process of positive factors during heterochronic parabiosis, there were limited understandings because the transportation process is nanoscale, which makes it difficult to identify the messenger unit. We demonstrated that heterochronic parabiosis could modulate HD in R6/2 mice model, and identified the messenger unit for transferring positive factors in the young blood serum. Methods: R6/2 mice were surgically connected with young wild-type mice (n = 13), old wild-type mice (n = 8), or R6/2 mice (n = 6) to examine the effect of heterochronic parabiosis. Parabionts composed of 5- to 6-week-old transgenic and wild-type mice were observed for 6 weeks in a single cage. The in vitro cellular model of HD cells were treated by the blood serum of the young or old mice, and by the exosomes isolated from thereof. The in vitro cellular model of HD were developed by differentiating neural stem cells cultured from SVZ of the brain. Results: After the heterochronic parabiosis, the weight loss and survival of HD mice was improved. Also, mutant Huntingtin aggregation (EM48 p

Reducing excessive inflammation is beneficial for the recovery from intracerebral hemorrhage (ICH). Here, the roles and mechanisms of A20 (TNFAIP3), an important endogenous anti-inflammatory factor, are examined in ICH. A20 expression in the PBMCs of ICH patients and an ICH mouse model was detected, and the correlation between A20 expression and neurologic deficits was analyzed. A20 expression was increased in PBMCs and was negatively related to the modified Rankin Scale score. A20 expression was also increased in mouse perihematomal tissues. A20−/− and A20-overexpressing mice were generated to further analyze A20 function. Compared with wild-type (WT) mice, A20−/− and A20-overexpressing mice showed significant increases and decreases, respectively, in hematoma volume, neurologic deficit score, mortality, neuronal degeneration, and proinflammatory factors. Moreover, WT-A20−/− parabiosis was established to explore the role of A20 in peripheral blood in ICH injury. ICH-induced damage, including brain edema, neurologic deficit score, proinflammatory factors, and neuronal apoptosis, was reduced in A20−/− parabionts compared with A20−/− mice. Finally, the interactions between TRAF6 and Ubc13 and UbcH5c were increased in A20−/− mice compared with WT mice; the opposite occurred in A20-overexpressing mice. Enhanced IκBα degradation and NF-κB activation were observed in A20−/− mice, but the results were reversed in A20-overexpressing mice. These results suggested that A20 is involved in regulating ICH-induced inflammatory injury in both the central and peripheral system and that A20 reduces ICH-induced inflammation by regulating TRAF6 polyubiquitination. Targeting A20 may thus be a promising therapeutic strategy for ICH.

Tissue-resident macrophages display varying phenotypic and functional properties that are largely specified by their local environment. One of these functions, phagocytosis, mediates the natural disposal of billions of cells, but its mechanisms and consequences within living tissues are poorly defined. Using a parabiosis-based strategy, we identified and isolated macrophages from multiple tissues as they phagocytosed blood-borne cellular material. Phagocytosis was circadianally regulated and mediated by distinct repertoires of receptors, opsonins, and transcription factors in macrophages from each tissue. Although the tissue of residence defined the core signature of macrophages, phagocytosis imprinted a distinct antiinflammatory profile. Phagocytic macrophages expressed CD206, displayed blunted expression of Il1b, and supported tissue homeostasis. Thus, phagocytosis is a source of macrophage heterogeneity that acts together with tissue-derived factors to preserve homeostasis., This study was supported by Ministerio de Economia, Competitividad e Industria (MINECO) grants SAF2015-65607 and SAF2013-49662-EXP to A. Hidalgo; SVP-2014-068595 to J.A. Nicolás-Ávila; BES-2013–065550 to J.M. Adrover; JCI-2012-12659 to N. A-Gonzalez; and SAF2014-56819-R and SAF2015-71878-REDT to A. Castrillo. Grant CAM S2010/BMD-2350 RAPHYME to A. Castrillo; SAF2015-64287-R, SAF2015-71878-REDT (MINECO), and CardioNext-ITN-608027 (European Commission FP7) to M. Ricote; and R01 AI089824 (National Institutes of Health) to C.V. Rothlin. H.P. is supported by grants from MINECO (SAF2014-54541-R), ATRES-MEDIA – AXA, Asociación Española Contra el Cáncer, WHRI Academy, and Worldwide Cancer Research. The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).

Aging is associated with a decline of various body functions, including ability to regenerate. Over recent decades, it has been demonstrated that some of these changes could be reversed in response to factors originating from a young organism, for example, fetal stem cells or "young blood" in models of heterochronic parabiosis. Pregnancy might be considered as parabiotic model of the interaction between two organisms of different age. In this work, we analyzed and summarized data on the effects of pregnancy on the maternal organism that confirm the hypothesis that pregnancy rejuvenates the mother's organism or slows its aging.

Background Inflammation underlies many forms of pulmonary hypertension ( PH ), including that resulting from Schistosoma infection, a major cause of PH worldwide. Schistosomiasis‐associated PH is proximately triggered by embolization of parasite eggs into the lungs, resulting in localized type 2 inflammation. However, the role of CD 4 + T cells in this disease is not well defined. Methods and Results We used a mouse model of schistosomiasis‐associated PH , induced by intraperitoneal egg sensitization followed by intravenous egg challenge, with outcomes including right ventricle systolic pressure measured by cardiac catheterization, and cell density and phenotype assessed by flow cytometry. We identified that embolization of Schistosoma eggs into lungs of egg‐sensitized mice increased the perivascular density of T‐helper 2 (Th2) CD 4 + T cells by recruitment of cells from the circulation and triggered type 2 inflammation. Parabiosis confirmed that egg embolization is required for localized type 2 immunity. We found Th2 CD 4 + T cells were necessary for Schistosoma ‐induced PH , given that deletion of CD 4 + T cells or inhibiting their Th2 function protected against type 2 inflammation and PH following Schistosoma exposure. We also observed that adoptive transfer of Schistosoma ‐sensitized CD 4 + Th2 cells was sufficient to drive type 2 inflammation and PH . Conclusions Th2 CD 4 + T cells are a necessary and sufficient component for the type 2 inflammation‐induced PH following Schistosoma exposure.

Age is a well-established risk factor for impaired bone fracture healing. Here, we identify a role for apolipoprotein E (ApoE) in age-associated impairment of bone fracture healing and osteoblast differentiation, and we investigate the mechanism by which ApoE alters these processes. We identified that, in both humans and mice, circulating ApoE levels increase with age. We assessed bone healing in WT and ApoE(–/–) mice after performing tibial fracture surgery: bone deposition was higher within fracture calluses from ApoE(–/–) mice. In vitro recombinant ApoE (rApoE) treatment of differentiating osteoblasts decreased cellular differentiation and matrix mineralization. Moreover, this rApoE treatment decreased osteoblast glycolytic activity while increasing lipid uptake and fatty acid oxidation. Using parabiosis models, we determined that circulating ApoE plays a strong inhibitory role in bone repair. Using an adeno-associated virus–based siRNA system, we decreased circulating ApoE levels in 24-month-old mice and demonstrated that, as a result, fracture calluses from these aged mice displayed enhanced bone deposition and mechanical strength. Our results demonstrate that circulating ApoE as an aging factor inhibits bone fracture healing by altering osteoblast metabolism, thereby identifying ApoE as a new therapeutic target for improving bone repair in the elderly.

OBJECTIVE To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.

Introduction: A loss of endogenous stem cells capable of tissue repair and regeneration drives the biological process that we recognize as "aging". Recovery of stem cell-mediated repair and regenerative functions in aged animals has been reported in murine heterochronic parabiosis experiments. Objectives: Herein we will review how pregnancy is an unusual form of heterochronic parabiosis, as the placenta prevents the exchange of most blood cells between parabionts. Instead, plasma and its content, including small extracellular vesicles, can readily cross the placental barrier. These nanosized extracellular vesicles are readily produced by the placenta, amnion, fetus and mother, and are essential for fetal organogenesis, growth and the progression of a healthy pregnancy. If defective, these extracellular vesicles can cause havoc such as in the case of peripartum cardiomyopathy. We will also review how these extracellular vesicles impact the mother substantially (including cardiac function) in the parabiosis of pregnancy. Conclusion: Extracellular vesicles generated during the course of a healthy pregnancy are essential for organogenesis and fetal growth, and also for maternal tissue repair and regeneration, and might be defective or deficient in pregnancies that result in peripartum cardiomyopathy.

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4(-/-)Tg). Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4(-/-)Tg mice with established disease at 8-9 weeks of age with C57BL/6 control mice. Such parabiotic "twins" had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4(-/-)Tg and CD8(-/-) mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8(+) T effector cells in the presence of wild type CD4 cells was noted. In conclusion, "correcting" the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.

Fat transplants increase body fat mass without changing the energy status of an animal and provide a tool for investigating control of total body fat. Early transplant studies found that small pieces of transplanted fat took on the morphology of the transplant recipient. Experiments described here tested whether this response was dependent upon expression of leptin receptors in either transplanted fat or the recipient mouse. Fat from leptin receptor deficient db/db mice or wild-type mice was placed subcutaneously in db/db mice. After 12 wk, cell size distribution in the transplant was the same as in endogenous fat of the recipient. Thus, wild-type fat cells, which express leptin receptors, were enlarged in a hyperleptinemic environment, indicating that leptin does not directly control adipocyte size. By contrast, db/db or wild-type fat transplanted into wild-type mice decreased in size, suggesting that a functional leptin system in the recipient is required for body fat mass to be controlled. In the final experiment, wild-type fat was transplanted into a db/db mouse parabiosed to either another db/db mouse to an ob/ob mouse or in control pairs in which both parabionts were ob/ob mice. Transplants increased in size in db/db–db/db pairs, decreased in db/db–ob/ob pairs and did not change in ob/ob-ob/ob pairs. We propose that leptin from db/db parabionts activated leptin receptors in their ob/ob partners. This, in turn, stimulated release of unidentified circulating factors, which travelled back to the db/db partner and acted on the transplant to reduce fat cell size.

The aim of this study was to investigate the consequences of maternal overweight on cardiac development in offspring in infants (short term) and minipigs (short and longer term).The epidemic of overweight involves pregnant women. The uterine environment affects organ development, modulating disease susceptibility. Offspring of obese mothers have higher rates of cardiovascular events and mortality.Echocardiography was performed in infants born to lean and overweight mothers at birth and at 3, 6, and 12 months of age. In minipigs born to mothers fed a high-fat diet or a normal diet, cardiac development (echocardiography, histology), glucose metabolism and perfusion (positron emission tomography), triglyceride and glycogen content, and myocardial enzymes regulating metabolism (mass spectrometry) were determined from birth to adulthood.In neonates, maternal overweight, especially in the last trimester, predicted a thicker left ventricular posterior wall at birth (4.1 ± 0.3 vs. 3.3 ± 0.2 mm; p 0.05) and larger end-diastolic and stroke volumes at 1 year. Minipigs born to mothers fed a high-fat diet showed greater left ventricular mass (p = 0.0001), chambers (+100%; p 0.001), stroke volume (+75%; p = 0.001), cardiomyocyte nuclei (+28%; p = 0.02), glucose uptake, and glycogen accumulation at birth (+100%; p 0.005), with lower levels of oxidative enzymes, compared with those born to mothers fed a normal diet. Subsequently, they developed myocardial insulin resistance and glycogen depletion. Late adulthood showed elevated heart rate (111 ± 5 vs. 84 ± 8 beats/min; p 0.05) and ejection fraction and deficient fatty acid oxidative enzymes.Neonatal changes in cardiac morphology were explained by late-trimester maternal body mass index; myocardial glucose overexposure seen in minipigs can justify early human findings. Longer term effects in minipigs consisted of myocardial insulin resistance, enzymatic alterations, and hyperdynamic systolic function.

Natural killer (NK) cells accumulate in adult murine and human uteri during decidualization induced physiologically, pathologically or experimentally. Adoptive transfer studies indicate that uterine NK (uNK) cells arise from circulating progenitors. However, virgin uteri contain few circulating NK1.1(+)CD49a(−) conventional NK (cNK) cells while NK1.1(+)CD49a(+) tissue-resident NK (trNK) cells are abundant. Here, we employed a novel, immune competent NK cell-specific reporter mouse to track accumulation of uNK cells during unmanipulated pregnancies. We identified cNK and trNK cells accumulating in both decidua basalis (DB) and myometrium. Only trNK cells showed evidence of proliferation. In parabiosis studies using experimentally-induced deciduomata, the accumulated uNK cells were proliferating trNK cells; migrating NK cells made no contribution. Together, these data suggest proliferating trNK cells are the source of uNK cells during endometrial decidualization.

Summary: Lung-resident primary memory CD8+ T cell populations (Trm) induced by a single influenza infection decline within months, rendering the host susceptible to new heterosubtypic influenza infections. Here, we demonstrate that, relative to single virus exposure, repeated antigen exposure dramatically alters the dynamics of influenza-specific lung Trm populations. Lung Trm derived from repeatedly stimulated circulating memory CD8+ T cells exhibit extended durability and protective heterosubtypic immunity relative to primary lung Trm. Parabiosis studies reveal that the enhanced durability of lung Trm after multiple antigen encounters resulted from the generation of long-lasting circulating effector memory (Tem) populations, which maintained the ability to be recruited to the lung parenchyma and converted to Trm, in combination with enhanced survival of these cells in the lung. Thus, generating a long-lasting Trm precursor pool through repeated intranasal immunizations might be a promising strategy to establish long-lasting lung Trm-mediated heterosubtypic immunity against influenza. : Van Braeckel-Budimir et al. find that repeated antigen exposure by influenza virus infection in the lung enhances the durability of lung CD8+ T resident memory populations and extends the duration of heterosubtypic immunity against influenza virus. Keywords: CD8+ T resident memory, lung, influenza, heterosubtypic protection

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma. In addition to disseminating through the cerebrospinal fluid, medulloblastoma can metastasize through circulating tumor cells in the blood, thereby providing a different angle for clinical diagnosis and treatment development.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

Fibrosis is the common final pathway of virtually all chronic injury to the kidney. While it is well accepted that myofibroblasts are the scar-producing cells in the kidney, their cellular origin is still hotly debated. The relative contribution of proximal tubular epithelium and circulating cells, including mesenchymal stem cells, macrophages, and fibrocytes, to the myofibroblast pool remains highly controversial. Using inducible genetic fate tracing of proximal tubular epithelium, we confirm that the proximal tubule does not contribute to the myofibroblast pool. However, in parabiosis models in which one parabiont is genetically labeled and the other is unlabeled and undergoes kidney fibrosis, we demonstrate that a small fraction of genetically labeled renal myofibroblasts derive from the circulation. Single-cell RNA sequencing confirms this finding but indicates that these cells are circulating monocytes, express few extracellular matrix or other myofibroblast genes, and express many proinflammatory cytokines. We conclude that this small circulating myofibroblast progenitor population contributes to renal fibrosis by paracrine rather than direct mechanisms.

Conquering obesity has become a major socioeconomic challenge. Here, we show that reduced expression of the miR-25-93-106b cluster, or miR-93 alone, increases fat mass and, subsequently, insulin resistance. Mechanistically, we discovered an intricate interplay between enhanced adipocyte precursor turnover and increased adipogenesis. First, miR-93 controls Tbx3, thereby limiting self-renewal in early adipocyte precursors. Second, miR-93 inhibits the metabolic target Sirt7, which we identified as a major driver of in vivo adipogenesis via induction of differentiation and maturation of early adipocyte precursors. Using mouse parabiosis, obesity in mir-25-93-106b(-/-) mice could be rescued by restoring levels of circulating miRNA and subsequent inhibition of Tbx3 and Sirt7. Downregulation of miR-93 also occurred in obese ob/ob mice, and this phenocopy of mir-25-93-106b(-/-) was partially reversible with injection of miR-93 mimics. Our data establish miR-93 as a negative regulator of adipogenesis and a potential therapeutic option for obesity and the metabolic syndrome. We graciously thank Flor Diaz for expert technical assistance. We are grateful to Andrea Ventura for providing the mir-25-93-106b-/- mice and Elena Lopez-Guadamillas for providing visceral fad pads from ob/ob mice. Research was supported by the European Research Council Advanced Investigator Grant (Pa-CSC 233460) and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreements 256974 (EPC-TM-NET) and 602783 (CAM-PaC). M.C. was supported by a La Caixa Fellowship. Sí

Age-related systemic environments influence neurogenesis and organ regeneration of heterochronic parabiotic partners; however, the difficulty of manipulating small embryos prevents the effects of aged systemic environments on primitive organs at the developmental stage from being analysed. Here, we describe a novel transplantation system to support whole living embryos/larvae as grafts in immunodeficient zebrafish by the intrusion of host blood vessels into the grafts, allowing bodies similar to those of heterochronic parabiosis to be generated by subcutaneous grafting. Although grafted embryos/larvae formed most organs, not all organogenesis was supported equally; although the brain, eyes and the intestine usually developed, the liver, testes and heart developed insufficiently or even occasionally disappeared. Removal of host germ cells stimulated testis development in grafted embryos. These results indicate that primitive testes are susceptible to the systemic environments that originated from the germ cells of aged hosts and imply that the primitive liver and heart are similar. Upon applying this method to embryonic lethal mutants, various types of organs, including testes that developed in germ-cell-removed recipients, and viable offspring were obtained from the mutants. This unique transplantation system will lead to new insights into the age-related systemic environments that are crucial for organogenesis in vertebrates.

Despite decades of intensive research, no drugs can cure or even stabilize Alzheimer's disease (AD). Current pharmacological treatments only partially mask the symptoms while the disease progresses within the brain. Finding a preventive measure or a cure for people with AD is indeed a worldwide urgent priority. A recent interesting study by T. Wyss-Coray's research group provides the first evidence that exposure to young blood or plasma can reverse some AD-related molecular and behavioral alterations. Heterochronic parabiosis (shared blood circulation) of AD transgenic mice with young healthy mice did not reduce amyloidosis and microglial activation in AD mice, but reversed the loss of synaptophysin and calbindin (critical synaptic proteins, indicators of cognitive decline in AD) in the dentate gyrus, and the abnormal expression, in the hippocampus, of many genes involved in key neuronal signaling pathways. Moreover, repeated intravenous administration of plasma from young healthy mice to AD mice reversed the excessive phosphorylation of hippocampal extracellular signal-regulated kinase (ERK), and improved spatial working memory and associative memory. Although observations in mouse models of AD might not necessarily extrapolate to humans, this preclinical study provides the first demonstration that young plasma has potential therapeutic properties, by ameliorating aspects of the disease that are present in AD patients. Clinical trials are already under way. If young plasma transfusion will be effective in AD patients, it will be important to identify the key factors responsible for the positive effects, as they might lead to the development of molecule interventions with a better efficacy/risk profile.

Green fluorescent protein is widely used in biological studies including parabiosis models for visualization of cellular structures and cells. However, the growing number of the data is available regarding immunogenicity of this protein, which can interfere with its use in in vivo experiments. In this study, we attempted to induce neonatal immunological tolerance to GFP-labeled karyocytes by intraperitoneal injections of B6.GFP mouse splenocytes to newborn C57/B6 mice. GFP+ skin graft integrity was evaluated under UV light at 6weeks after skin grafting. GFP+ skin transplants survived up to 6weeks after grafting in all animals that undergone neonatal tolerance induction, whereas all skin grafts were rejected in control naive mice within first two weeks. Thus, current protocol is suitable for induction of immune tolerance against GFP-labeled karyocytes.