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Alloantibodies in presensitized transplant candidates deposit complement membrane attack complexes (MACs) on graft endothelial cells (ECs), increasing risk of CD8(+) T cell–mediated acute rejection. We recently showed that human ECs endocytose MACs into Rab5(+) endosomes, creating a signaling platform that stabilizes NF-κB–inducing kinase (NIK) protein. Endosomal NIK activates both noncanonical NF-κB signaling to synthesize pro–IL-1β and an NLRP3 inflammasome to process and secrete active IL-1β. IL-1β activates ECs, increasing recruitment and activation of alloreactive effector memory CD4(+) T (Tem) cells. Here, we report that IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1β stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB–dependent process in which IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8(+) Tem cells. Blocking NLRP3 inflammasome assembly, IL-1 receptor, or IL-15 on ECs inhibited the augmented CD8(+) Tem cell responses, indicating that this pathway is not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice, and enhanced intimal CD8(+) T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete. Impact Statement Three Dimensional printing can be used to generate multilayered vascularized human skin grafts that can potentially overcome the limitations of graft survival observed in current avascular skin substitutes. Inclusion of human pericytes in the dermal bioink appears to improve both dermal and epidermal maturation.

Rationale: Complement activation contributes to multiple immune-mediated pathologies. In late allograft failure, donor-specific antibody deposits complement membrane attack complexes (MAC) on graft endothelial cells (ECs), substantially increasing their immunogenicity without causing lysis. Internalized MAC stabilize NIK (NF-κB [nuclear factor kappa-light-chain-enhancer of activated B cells]–inducing kinase) protein on Rab5+MAC+ endosomes, activating noncanonical NF-κB signaling. However, the link to increased immunogenicity is unclear. Objective: To identify mechanisms by which alloantibody and internalized MAC activate ECs to enhance their ability to increase T-cell responses. Methods and Results: In human EC cultures, internalized MAC also causes NLRP3 (NOD-like receptor family pyrin domain containing 3) translocation from endoplasmic reticulum to Rab5+MAC+NIK+ endosomes followed by endosomal NIK-dependent inflammasome assembly. Cytosolic NIK, stabilized by LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells), does not trigger inflammasome assembly, and ATP-triggered inflammasome assembly does not require NIK. IFN-γ (interferon-γ) primes EC responsiveness to MAC by increasing NLRP3, pro-caspase 1, and gasdermin D expression. NIK-activated noncanonical NF-κB signaling induces pro-IL (interleukin)-1β expression. Inflammasome processed pro-IL-1β, and gasdermin D results in IL-1β secretion that increases EC immunogenicity through IL-1 receptor signaling. Activation of human ECs lining human coronary artery grafts in immunodeficient mouse hosts by alloantibody and complement similarly depends on assembly of an NLRP3 inflammasome. Finally, in renal allograft biopsies showing chronic rejection, caspase-1 is activated in C4d + ECs of interstitial microvessels, supporting the relevance of the cell culture findings. Conclusions: In response to antibody-mediated complement activation, IFN-γ-primed human ECs internalize MAC, triggering both endosomal-associated NIK-dependent NLRP3 inflammasome assembly and IL-1 synthesis, resulting in autocrine/paracrine IL-1β-mediated increases in EC immunogenicity. Similar responses may underlie other complement-mediated pathologies.

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions., Complement activation contributes to vascular inflammation in the contexts of allograft rejection and connective tissue disease. Here Fang et al. identify ZFYVE21 as a novel effector of Rab5 and find it regulates pro-inflammatory NF-κB signaling in endothelial cells in response to complement activation.

Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of β(2)-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4(+) effector memory T cells and are resistant to killing by CD8(+) alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell–cell interactions of circulating leukocytes with IL-1– or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17–induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17–activated PCs, but not ECs, induce pertussis toxin–sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17–activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17–treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17–activated PCs can modulate neutrophil functions within the perivascular tissue space.

Manipulation of human T cell functioning by delivery of macromolecules such as DNA, RNA, or protein is limited, unless the human T cells have been stimulated or electropermeabilized. To achieve successful adaptation and survival of a grafted organ, the alloreactive T cells that induce graft rejection must be regulated. Corticosteroids, calcineurin inhibitors, and mTOR inhibitors, which are systemic immunosuppressants, are currently used for transplantation, with significant side effects. In this study, we demonstrated that a cell-permeable peptide (CPP), dNP2, could efficiently deliver proteins into human CD4 and CD8 T cells. We confirmed regulatory functioning of the cytoplasmic domain of CTLA-4 conjugated with dNP2 (dNP2-ctCTLA-4) in human T cell activation, proliferation, and chemokine receptor expression. We utilized a human skin allograft system in SCID/beige mice to examine whether dNP2-ctCTLA-4 could inhibit allograft rejection by controlling T cell responses. The grafted skin tissue inflammation, allogeneic T cell infiltration, and blood cytokine level was markedly reduced by dNP2-ctCTLA4, resulting in successful transplantation. In addition, it also inhibited T cell alloresponses against microvessels formed form Bcl-2-transduced human umbilical vein endothelial cells implanted into Balb/c Rag1(−/−)/IL-2Rγ(−/−) double knockout (DKO) mice, assessed as reduced T cell infiltration and granzyme B expression. These results collectively suggest that dNP2 peptide conjugation offers a valuable tool for delivering macromolecules like proteins into human T cells, and dNP2-ctCTLA-4 is a novel agent that shows potential in controlling human T cell responses to allow successful adaptation of grafted tissues.

Early acute rejection of human allografts is mediated by circulating alloreactive host effector memory T cells (TEM). TEM infiltration typically occurs across graft postcapillary venules and involves sequential interactions with graft-derived endothelial cells (ECs) and pericytes (PCs). While the role of ECs in allograft rejection has been extensively studied, contributions of PCs to this process are largely unknown. This study aimed to characterize the effects and mechanisms of interactions between human PCs and allogeneic TEM. We report that unstimulated PCs, like ECs, can directly present alloantigen to TEM, but while IFN-γ-activated ECs (γ-ECs) show increased ability to stimulate alloreactive T cells, IFN-γ-activated PCs (γ-PCs) instead suppress TEM proliferation but not cytokine production or signaling. RNA sequencing analysis of PCs, γ-PCs, ECs, and γ-ECs reveal induction of indoleamine 2,3-dioxygenase 1 (IDO1) in γ-PCs to significantly higher levels than in γ-ECs that correlates with tryptophan depletion in vitro. Consistently, shRNA knockdown of IDO1 markedly reduces γ-PC-mediated immunoregulatory effects. Furthermore, human PCs express IDO1 in a skin allograft rejection humanized mouse model and in human renal allografts with acute T cell-mediated rejection. We conclude that immunosuppressive properties of human PCs are not intrinsic but instead result from IFN-γ-induced IDO1-mediated tryptophan depletion.

Ex vivo normothermic machine perfusion (NMP) is a new clinical strategy to assess and resuscitate organs likely to be declined for transplantation, thereby increasing the number of viable organs available. Short periods of NMP provide a window of opportunity to deliver therapeutics directly to the organ and, in particular, to the vascular endothelial cells (ECs) that constitute the first point of contact with the recipient’s immune system. ECs are the primary targets of both ischemia-reperfusion injury and damage from preformed antidonor antibodies, and reduction of perioperative EC injury could have long-term benefits by reducing the intensity of the host’s alloimmune response. Using NMP to administer therapeutics directly to the graft avoids many of the limitations associated with systemic drug delivery. We have previously shown that polymeric nanoparticles (NPs) can serve as depots for long-term drug release, but ensuring robust NP accumulation within a target cell type (graft ECs in this case) remains a fundamental challenge of nanomedicine. We show that surface conjugation of an anti-CD31 antibody enhances targeting of NPs to graft ECs of human kidneys undergoing NMP. Using a two-color quantitative microscopy approach, we demonstrate that targeting can enhance EC accumulation by about 5-to 10-fold or higher in discrete regions of the renal vasculature. In addition, our studies reveal that NPs can also non-specifically accumulate within obstructed regions of the vasculature that are poorly perfused. These quantitative preclinical human studies demonstrate the therapeutic potential for targeted nanomedicines delivered during ex vivo NMP.

Rapamycin modulates the ability of the vascular endothelium to mediate inflammation by inhibiting mTORC2 and reducing TNF-induced VCAM-1 expression., Recruitment of circulating leukocytes into inflamed tissues depends on adhesion molecules expressed by endothelial cells (ECs). Here we report that rapamycin pretreatment reduced the ability of TNF-treated ECs to capture T cells under conditions of venular flow. This functional change was caused by inhibition of TNF-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and could be mimicked by knockdown of mammalian target of rapamycin (mTOR) or rictor, but not raptor, implicating mTORC2 as the target of rapamycin for this effect. Mechanistically, mTORC2 acts through Akt to repress Raf1-MEK1/2-ERK1/2 signaling, and inhibition of mTORC2 consequently results in hyperactivation of ERK1/2. Increased ERK1/2 activity antagonizes VCAM-1 expression by repressing TNF induction of the transcription factor IRF-1. Preventing activation of ERK1/2 reduced the ability of rapamycin to inhibit TNF-induced VCAM-1 expression. In vivo, rapamycin inhibited mTORC2 activity and potentiated activation of ERK1/2. These changes correlated with reduced endothelial expression of TNF-induced VCAM-1, which was restored via pharmacological inhibition of ERK1/2. Functionally, rapamycin reduced infiltration of leukocytes into renal glomeruli, an effect which was partially reversed by inhibition of ERK1/2. These data demonstrate a novel mechanism by which rapamycin modulates the ability of vascular endothelium to mediate inflammation and identifies endothelial mTORC2 as a potential therapeutic target.

We hypothesized that de novo donor-specific antibody (DSA) causes complement-dependent endothelial cell injury in kidney transplants, as assessed by expression of endothelial cell-associated transcripts (ENDATs), that may be attenuated through complement inhibition. In total, 15 participants (five control, 10 treatment) with DSA and deteriorating renal function were enrolled. The treatment group received 6 mo of eculizumab followed by 6 mo of observation, whereas controls were observed. The primary end point was percentage change in estimated GFR (eGFR) trajectory over the treatment period. The treatment group had an improved eGFR trajectory versus control, based on our predetermined two-sided 0.10 significance level (p = 0.09). Within-subject analysis of treated participants at 6-mo intervals did not show significant change (p = 0.60). Modeling C1q status showed that C1q-positive patients had significantly higher mean eGFR than patients with negative C1q (p = 0.04). Biopsies revealed elevated renal ENDATs in most participants, but ENDATs were not reduced with complement inhibition. Our data suggest that eculizumab treatment may stabilize kidney function in patients with chronic persistent DSA based on our pilot a priori significance threshold. ENDAT expression predicative of acute humoral injury is not reduced with complement inhibition in this chronic setting. Further studies will be necessary to determine which patients may benefit from eculizumab.

Objective— Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. Methods and Results— Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to “heal in” for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell–mediated injury to human artery segments. Conclusion— We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Allograft vasculopathy (AV) is characterized by diffuse stenoses in the vasculature of solid organ transplants. Previously, we developed two humanized models showing that alloantibody and ischemia reperfusion injury (IRI) exacerbated T cell-mediated AV in human arterial xenografts in vivo. Herein we examined a causal role for terminal complement activation in both settings. IRI, in contrast to alloantibody, elicited widespread membrane attack complex (MAC) assembly throughout the vessel wall. Both alloantibody and IRI caused early (24 h) and robust endothelial cell (EC) activation localized to regions of intimal MAC deposition, indicated by increases in nuclear factor kappa B (NF-κB)-inducing kinase, an MAC-dependent activator of noncanonical NF-kB, VCAM-1 expression and Gr-1+ neutrophil infiltration. Endothelial cell activation by alloantibody was inhibited by antimouse C5 mAb, but not by anti-C5a mAb or by control mAb, implicating MAC as the primary target of anti-C5 mAb. Antimouse C5 mAb significantly reduced alloantibody- and IRI-enhanced T cell infiltration and AV-like changes, including neointimal hyperplasia as well as intraluminal thrombosis in a subset of IRI-treated arterial grafts. These results indicate that increased AV lesion formation in response to either alloantibody or IRI is dependent on complement C5 activation and, accordingly, inhibition of this pathway may attenuate AV.

Interleukin (IL) 1α produced by human endothelial cells (ECs), in response to tumor necrosis factor (TNF) or to co-culture with allogeneic T cells in a TNF-dependent manner, can augment the release of cytokines from alloreactive memory T cells in vitro. In a human–mouse chimeric model of artery allograft rejection, ECs lining the transplanted human arteries express IL-1α, and blocking IL-1 reduces the extent of human T cell infiltration into the artery intima and selectively inhibits IL-17 production by infiltrating T cells. In human skin grafts implanted on immunodeficient mice, administration of IL-17 is sufficient to induce mild inflammation. In cultured cells, IL-17 acts preferentially on vascular smooth muscle cells rather than ECs to enhance production of proinflammatory mediators, including IL-6, CXCL8, and CCL20. Neutralization of IL-17 does not reduce T cell infiltration into allogeneic human artery grafts, but markedly reduces IL-6, CXCL8, and CCL20 expression and selectively inhibits CCR6+ T cell accumulation in rejecting arteries. We conclude that graft-derived IL-1 can promote T cell intimal recruitment and IL-17 production during human artery allograft rejection, and suggest that targeting IL-1 in the perioperative transplant period may modulate host alloreactivity.

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (TEM) and central memory (TCM) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ TEM (CD45RO+CCR7−CD62L−) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ TCM (CD45RO+CCR7+CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ TEM secrete more IFN-γ within 48 h in response to allogeneic ECs than do TCM. In contrast, TEM and TCM secrete comparable amounts of IFN-γ in response to allogeneic monocytes (Mo). In the same cultures, both TEM and TCM produce IL-2 and proliferate in response to IFN-γ-treated allogeneic human EC or Mo, but TCM respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-γ secretion by CD4+ TEM cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-γ production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic TEM production of IL-2 but not IFN-γ. We conclude that human CD4+ TEM directly recognize and respond to allogeneic EC in vitro by secreting IFN-γ and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ TEM can mediate allogeneic EC injury in vivo.

Although antibodies to HLA play a role in the pathogenesis of diseases processes such as rejection of transplanted organs, the precise mechanisms by which antibodies cause tissue injury are not completely understood. We hypothesized that antibodies to host tissues cause inflammation in part by activating endothelial exocytosis of granules that contain prothrombotic mediators such as von Willebrand Factor (VWF) and proinflammatory mediators such as P-selectin. To test this hypothesis, we treated human endothelial cells with murine monoclonal antibody W6/32 to HLA class I and then measured exocytosis by the release of VWF and the externalization of P-selectin. Antibody to HLA activates endothelial exocytosis in a dose-dependent manner over time. The biologically active complement split product, C5a, adds a slight but significant increase to antibody induction of exocytosis. Antibody to HLA alone or with C5a did not damage the cells. Cross-linking of HLA appears to play a role in the ability of antibody to activate exocytosis, because the W6/32 monovalent Fab fragment did not activate VWF release, but the bivalent F(ab′) 2 was effective in triggering exocytosis. To explore the in vivo effects of antibody upon graft injury, we infused W6/32 F(ab′) 2 antibody to human HLA into severe combined immunodeficient/beige mice that had been transplanted with human skin grafts. Antibody to HLA activated exocytosis and inflammation in human skin grafts. Our data show that antibody to host antigens can activate human endothelial cell exocytosis and leukocyte trafficking. By triggering vascular inflammation, antibody activation of exocytosis may play a role in transplant rejection.

Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB-inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5(+)endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC(+) endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt(+)NIK(+) signalosome on Rab5(+) endosomes.

IL-11 can reduce tissue injury in animal models of inflammation but the mechanism(s) is unknown. When C.B-17 SCID/beige mice bearing human skin grafts are injected i.p. with human PBMC allogeneic to the donor skin, infiltrating T cells destroy human microvessels by day 21. Intradermal injection of human IL-11 (500 ng/day) delays the time course of graft microvessel loss without reducing the extent of T cell infiltration. Protective actions of IL-11 are most pronounced on day 15. IL-11 has no effect on T cell activation marker, effector molecule, cytokine expression, or endothelial ICAM-1 expression. IL-11 up-regulates the expression of survivin, a cytoprotective protein, in graft keratinocytes and endothelial cells. Topical application of survivin antisense oligonucleotide down-regulates survivin expression in both cell types and largely abrogates the protective effect of IL-11. We conclude that in this human transplant model, IL-11 exerts a cytoprotective rather than anti-inflammatory or immunomodulatory effect mediated through induction of survivin.

BACKGROUND We have previously demonstrated that human artery grafts transplanted to immunodeficient mice are infiltrated and injured by unsensitized allogeneic human T cells. We extended our investigations to human anti-porcine xenoresponses in this model. METHODS Pig coronary artery segments were interposed into the infrarenal aorta of severe combined immunodeficiency/beige mice. After 7 days, certain recipients were reconstituted with human leukocytes and/or treated with proinflammatory cytokines. The grafts were harvested after 1-70 days and examined by histology, immunohistochemistry, and morphometry. RESULTS Pig artery grafts from untreated mice had no evidence of injury, leukocytic infiltrate, or endothelial cell activation up to 70 days postoperatively, despite deposition of murine complement. Host reconstitution with human peripheral blood mononuclear cells resulted in a discrete population of circulating T cells that did not infiltrate or injure the grafts up to 28 days after adoptive transfer. Administration of porcine interferon-gamma for up to 28 days sustained the expression of graft vascular cell adhesion molecule-1 and major histocompatibility complex antigens, but did not initiate recruitment of human leukocytes. In contrast, treatment with human tumor necrosis factor for 7 days induced the de novo expression of porcine E-selectin by graft endothelial cells and elicited human T cell infiltration and human peripheral blood mononuclear cell-dependent vascular injury. CONCLUSIONS The human peripheral blood mononuclear cell-severe combined immunodeficiency/beige mouse model identifies a significant difference between human T cell allogeneic and xenogeneic responses in vivo. Xenografts with quiescent endothelium are not infiltrated or injured by T cells under the same conditions in which allografts are rejected. Activation of pig coronary artery endothelial cells by human tumor necrosis factor, but not porcine interferon-gamma, elicits cellular xenoresponses.

Interleukin-11 (IL-11) reduces injury both in vivo and in vitro, but the mechanisms are unknown. Stimulation of serum- and growth factor-deprived HUVEC with IL-11 increased survivin mRNA and protein expression levels in a dose-dependent manner, with maximal induction at 50 to 100 ng/ml of IL-11. Survivin mRNA expression peaked after 3 to 6 hours of IL-11 treatment and decreased by 24 hours. Survivin protein expression was maximal at 6 hours of treatment and remained elevated through 24 hours. Survivin induction may be mediated by activation of protein kinase B/Akt, but IL-11 failed to activate this pathway in HUVEC. IL-11 did activate signal transducer and activator of transcription (STAT)-3 and IL-11 failed to induce survivin expression in HUVEC transduced with a dominant-negative STAT3 mutant, whereas control-transduced HUVEC responded normally. An IL-11 transgene caused increased survivin mRNA expression in mice compared with control littermates. Intradermal injection of IL-11 (500 ng) into human skin xenografts on immunodeficient mice up-regulated survivin protein in microvascular endothelium and epithelial keratinocytes. We conclude that IL-11 induces expression of survivin, an antiapoptotic protein, in vitro and in vivo, and identify STAT3 as a critical mediator of this response.

TNF activates endothelial cells to express cell surface molecules that are necessary to recruit a local infiltrate of leukocytes. Because the actions of this proinflammatory cytokine are not species restricted, we investigated whether human TNF can up-regulate porcine endothelial adhesion molecules to elicit human T cell infiltration and damage of pig skin xenografts in a chimeric immunodeficient mouse model. We have previously demonstrated the vigorous rejection of human skin allografts and the absence of injury to porcine skin xenografts in human PBMC-SCID/beige mice. Intradermal administration of human TNF at high doses (600 or 2000 ng) caused nonspecific inflammatory damage of pig skin grafts, whereas low concentrations of TNF (60 or 200 ng) resulted in human PBMC-dependent injury of porcine endothelial cells. There was a strong correlation among pig skin xenograft damage, human T cell infiltration, and the TNF-induced up-regulation of swine MHC class I and class II molecules, VCAM-1, and, in particular, the de novo expression of porcine E-selectin. The microvascular damage and leukocytic infiltration elicited by TNF were enhanced by porcine IFN-γ, suggesting that xenografts may be less prone to cytokine-mediated injury due to the species-restricted effects of recipient IFN-γ. Our results indicate that maintenance of a quiescent endothelium, which does not express E-selectin or other activation-dependent adhesion molecules, is important in preventing human anti-porcine T cell xenoresponses in vivo and that TNF signaling molecules and TNF-responsive gene products are appropriate therapeutic targets to protect against human T cell-mediated rejection of pig xenografts.

Background— Cardiac allograft vasculopathy is the major cause of late allograft loss after heart transplantation. Cardiac allograft vasculopathy lesions contain alloreactive T cells that secrete interferon-γ, a vasculopathic cytokine, and occur more frequently in patients with donor-specific antibody. Pathological interactions between these immune effectors, representing cellular and humoral immunity, respectively, remain largely unexplored. Methods and Results— We used human panel reactive antibody to form membrane attack complexes on allogeneic endothelial cells in vitro and in vivo. Rather than inducing cytolysis, membrane attack complexes upregulated inflammatory genes, enhancing the capacity of endothelial cells to recruit and activate allogeneic interferon-γ––producing CD4 + T cells in a manner dependent on the activation of noncanonical nuclear factor-κB signaling. Noncanonical nuclear factor-κB signaling was detected in situ within endothelial cells both in renal biopsies from transplantation patients with chronic antibody-mediated rejection and in panel-reactive antibody––treated human coronary artery xenografts in immunodeficient mice. On retransplantation into immunodeficient hosts engrafted with human T cells, panel-reactive antibody––treated grafts recruited more interferon-γ––producing T cells and enhanced cardiac allograft vasculopathy lesion formation. Conclusions— Alloantibody and complement deposition on graft endothelial cells activates noncanonical nuclear factor-κB signaling, initiating a proinflammatory gene program that enhances alloreactive T cell activation and development of cardiac allograft vasculopathy. Noncanonical nuclear factor-κB signaling in endothelial cells, observed in human allograft specimens and implicated in lesion pathogenesis, may represent a target for new pharmacotherapies to halt the progression of cardiac allograft vasculopathy.

OBJECTIVES: Shiga-toxin producing O157:H7 Entero Haemorrhagic E. coli (STEC/EHEC) is one of the most common causes of Haemolytic Uraemic Syndrome (HUS) related to infectious haemorrhagic colitis. Nearly all recommendations on clinical management of EHEC infections refer to this strain. The 2011 outbreak in Northern Europe was the first to be caused by the serotype O104:H4. This EHEC strain was found to carry genetic features of Entero Aggregative E. coli (EAEC) and extended spectrum β lactamase (ESBL). We report symptoms and complications in patients at one of the most affected centres of the 2011 EHEC O104 outbreak in Northern Germany. METHODS: The courses of patients admitted to our hospital due to bloody diarrhoea with suspected EHEC O104 infection were recorded prospectively. These data include the patients' histories, clinical findings, and complications. RESULTS: EHEC O104 infection was confirmed in 61 patients (female = 37; mean age: 44±2 years). The frequency of HUS was 59% (36/61) in our cohort. An enteric colonisation with co-pathogens was found in 57%. Thirty-one (51%) patients were treated with plasma-separation/plasmapheresis, 16 (26%) with haemodialysis, and 7 (11%) with Eculizumab. Patients receiving antibiotic treatment (n = 37; 61%) experienced no apparent change in their clinical course. Twenty-six (43%) patients suffered from neurological symptoms. One 83-year-old patient died due to comorbidities after HUS was successfully treated. CONCLUSIONS: EHEC O104:H4 infections differ markedly from earlier reports on O157:H7 induced enterocolitis in regard to epidemiology, symptomatology, and frequency of complications. We recommend a standard of practice for clinical monitoring and support the renaming of EHEC O104:H4 syndrome as "EAHEC disease".

The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

Microvascular endothelial cell (EC) expression of tumor necrosis factor receptor (TNFR) 2 is induced in situ by ischemia/reperfusion injury. To assess effects of molecular oxygen on TNFR2 expression, we subjected cultured human dermal microvascular ECs (HDMECs) to hypoxic conditions (1% O(2)) or to hypoxic conditions followed by return to normoxic conditions. TNFR2 mRNA and protein are expressed under normoxic conditions but are rapidly reduced by hypoxia; they fall even further upon reoxygenation but rebound by 6-9 h. TNFR1 expression is unaffected by hypoxia or reoxygenation in these same cells. We identified a potential FOXO3a binding site in the 5' enhancer region of the TNFR2 gene. FOXO3a from normoxic but not hypoxic HDMECs binds an oligonucleotide sequence matching this site, and the endogenous enhancer binds FOXO3a at this site in HDMECs under normoxic but not hypoxic conditions. Unphosphorylated FOXO3a is present in the nucleus of HDMECs under normoxic conditions. Hypoxia leads to FOXO3a phosphorylation at an Akt/protein kinase B target site and subsequent nuclear export; these processes are reversed by reoxygenation and blocked by LY294002, a phosphatidylinositol 3-kinase inhibitor that blocks Akt activation. LY294002 also prevents the hypoxia-mediated decrease in TNFR2 expression. Transiently transfected FOXO3a activates a TNFR2 promoter/reporter construct in HDMECs, whereas small interference RNA knockdown of FOXO3a reduces TNFR2 but not TNFR1 expression under normoxic conditions. Reduction in TNFR2 by small interference RNA sensitizes HDMECs to TNFR1-mediated apoptosis. We conclude that FOXO3a regulates oxygen-dependent changes in expression of TNFR2 in HDMECs, controlling sensitivity to TNF-mediated apoptosis.

Cell-mediated rejection responses of allogeneic organs often involve damage to graft blood vessels, especially of the endothelial cell (EC) lining (1, 2). The mechanisms underlying injury of allogeneic ECs in clinical transplantation are elusive for several reasons. First, commonly used rodent models may be misleading because human T-cell responses to allogeneic ECs differ in significant ways from those that occur in rodents. For example, rodent EC do not basally express major histocompatibility complex (MHC) class II molecules or the costimulator lymphocyte function-associated antigen-3, yet may express the costimulator CD86. In vitro, human EC can activate allogeneic CD4+ and CD8+ memory T cells through direct recognition of allogeneic MHC molecules whereas mouse EC cannot activate CD4+ effector cells (3). Second, observational and interventional studies of human transplant recipients are significantly limited by the inability to control experimental variables in clinical settings. Third, human cell culture experiments do not adequately approximate allograft rejection, an in vivo process. For these reasons, we have developed several models using C.B-17 severe combined immunodeficiency (SCID)/beige (bg) immunodeficient mouse hosts in which human memory T-cell responses to allogeneic skin or artery graft tissues can be studied in vivo (4–7). However, only T cells and, to a limited extent, B cells successfully engraft in these animals despite the presence of natural killer (NK) cells, monocytes, and dendritic cells in the initial inoculum (8). The models thus differ from rejecting clinical allografts in which macrophages, derived from blood monocytes, comprise as many as half of infiltrating leukocytes (9). Since mature human blood monocytes are not readily transferable into mouse hosts, we turned to human hematopoietic stem-cell (HSC) transplantation based on the knowledge that transplantation of mouse HSC into an irradiated autologous host can fully regenerate the hematopoietic system, including macrophages. However, immunocompetent mice rapidly reject human hematopoietic cells, including HSCs by a combination of innate and adaptive immune effector mechanisms. Compromise of the adaptive immune system of mice first spontaneously arose in C.B-17 animals by mutation in the Prkdc gene whose protein product participates in immunoglobulin (Ig) and T-cell receptor rearrangements (10). Animals bearing this trait are referred to as having the SCID mutation. Despite an essentially absent adaptive immune system, C.B-17 SCID animals are still significantly resistant to adoptive transfer of human peripheral blood lymphocyte (PBL). Host NK cells were proposed to play a major role in this resistance because of their known capacity to reject allogeneic hematopoietic cells (11). Consistent with this interpretation, we found that resistance to adoptive transfer of human PBL was reduced in animals treated with antibody to asialo GM1, which depletes NK cells, or in C.B-17 SCID animals that also have the bg mutation that impairs the function of NK cells (as well as that of granulocytes and platelets) (4–6). The SCID mutation was bred into nonobese diabetic (NOD) mice by others to study the role of the adaptive immune system in the autoimmune phenotypes to which wild-type NOD mice are subject (12). In addition to their propensity to develop autoimmune disease, NOD mice are also deficient for certain class I MHC-related genes that play a role in the positive selection of NK cells from their progenitors (13). Consequently, compared with C.B-17 SCID mice, NOD SCID animals are additionally deficient in mature NK cells. For this reason, NOD SCID animals are more accepting of adoptively transferred human PBL than are C.B-17 SCID animals (14). (It was unclear if NOD SCID animals are also more accepting of adoptively transferred human T cells than are C.B-17 SCID/bg mice since these two strains had not previously been compared.) Despite their acceptance of mature T cells, NOD SCID animals still resist transplantation of human HSCs, even when irradiated to further impair immunity and to establish empty bone marrow niches. An additional mutation, namely knock out of the common gamma chain of the cytokine receptor family (γc), was introduced into NOD SCID animals to further compromise innate effector cells, many of which depend on γc signaling cytokines for their development (14). The resultant NOD SCID γc−/− strain has been reported to be an excellent host for human HSCs, allowing significant reconstitution of a human hematopoietic and immune system by HSCs after irradiating. There have not, to the best of our knowledge, been systematic comparisons between irradiated C.B-17 SCID/bg and NOD SCID γc−/− animal as recipients of human HSCs. There is also little known about how these animal strains compare with regard to acceptance of vascularized human tissues that may be recognized by elements of the innate immune system other than NK cells. Indeed many aspects of innate mechanisms of xenoreactivity are still unappreciated. For example, it has only recently been shown that NOD animals contain an allelic form of the signal regulatory protein (SIRP)-α inhibitory receptor on their macrophages that can interact with a human CD47 counter-receptor whereas non-obese diabetes-resistant (NOR) mice express an allele that cannot (15). It is not known which allelic forms SIPR-α are expressed in C.B-17 mice. The key points are that innate immune responses of different mouse strains to xenogeneic human grafts are not yet predictable and may vary with the type of graft. In the present study, we directly compare the recently developed-NOD SCID γc−/− mouse strain(16), with SCID/bg animals as recipients of human skin, artery, or HSCs isolated from the peripheral blood of G-colony-stimulating factor (CSF)-mobilized adult donors. This source of HSCs allows us to combine stem-cell engraftment with adoptive transfer of alloreactive memory T cells that are important in allograft rejection (17). We find that SCID/bg adult animals offer major technical advantages over NOD SCID γc−/− as recipients of human skin and artery, whereas NOD SCID γc−/− mice much more readily accept human blood HSCs. However, human HSCs do give rise to mononuclear phagocytes in SCID/bg hosts and we use this model to show that human macrophages can actively contribute to experimental allograft rejection.

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.

Interferon (IFN)-gamma actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-gamma signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-gamma on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-gamma consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs. STAT3 activation resulted in nuclear translocation of this transcription factor. By selectively inhibiting STAT3 and not STAT1 signaling, we identified a number of candidate IFN-gamma-inducible, STAT3-dependent gene products by microarray analysis. Results for selected genes, including the pro-apoptotic molecules X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa, were verified by real time quantitative reverse transcription-PCR and immunoblot analyses. IFN-gamma-induced STAT3 and STAT1 signaling in VSMCs demonstrated reciprocal inhibition. STAT3 activation by IFN-gamma sensitized VSMCs to apoptosis triggered by both death receptor- and mitochondrial-mediated pathways. Knock down of XAF1 and Noxa expression inhibited the priming of VSMCs to apoptotic stimuli by IFN-gamma. Finally, we confirmed the in vivo relevance of our observations using a chimeric animal model of immunodeficient mice bearing human coronary artery grafts in which the expression of XAF1 and Noxa as well as the pro-apoptotic effects induced by IFN-gamma were dependent on STAT3. The data suggest STAT1-independent signaling by IFN-gamma via STAT3 that promotes the death of human VSMCs.

Background Secondary vasculitis represents a rare extraintestinal manifestation of Crohn's disease (CD). Appropriate and prompt diagnosis is often delayed by uncertainties about the relationship of the vasculitic manifestations and CD. Objective To describe our experience with vasculitis in CD and review the literature with respect to different manifestations and pathophysiological aspects of extraintestinal vasculitic manifestations of CD. Methods We report 2 new cases of CD with secondary small-vessel vasculitis. We also extensively review the literature (1960-2007) using a broad range of key words related to secondary vasculitis in CD. Relevant publications were evaluated for the number of reported patients and manifestations of vasculitis. Results Vasculitis is a rare extraintestinal manifestation of CD. Different types of vasculitis affect large-, medium-, and small-sized vessels associated with CD. Common immunologic features include intestinal inflammation as well as an infiltration of γδ-T-cells and/or Th1-type cells into vessel walls. The 2 new cases of secondary vasculitis in CD reported here reflect 2 major types of CD-related inflammatory vascular disorders. The first involves the central nervous system, while the second represents circumscribed Musculus gastrocnemius involvement (so-called "gastrocnemius myalgia syndrome"). Successful treatment of refractory secondary vasculitis in CD with an anti-tumor necrosis factor-α antibody is shown for the first time. Conclusion Vasculitis secondary to CD is an uncommon finding. Therefore, it has to be carefully differentiated from other forms of primary or secondary vasculitis with intestinal involvement. Treatment with an anti- tumor necrosis factor-α antibody may prove a treatment option in vasculitis as an extraintestinal manifestation of CD.

Human TRAIL can efficiently kill tumor cells in vitro and kill human tumor xenografts in mice with little effect on normal mouse cells or tissues. The effects of TRAIL on normal human tissues have not been described. In this study, we report that endothelial cells (EC), isolated from human umbilical veins or human dermal microvessels, express death domain-containing TRAIL-R1 and -R2. Incubation with TRAIL for 15 h causes ∼30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4′,6′-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-κB, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL.

Oncostatin M (OnM) signals through cell surface receptors, which utilize the gp130 subunit. In cultured human umbilical vein endothelial cells (HUVEC), OnM transiently elevates mRNA encoding for suppressor of cytokine signaling-3 (SOCS-3). By 1 h of OnM treatment, HUVEC become refractory to the restimulation by OnM, measured as failure to reinduce SOCS-3 mRNA. OnM-induced desensitization also prevents responses to other gp130-signaling cytokines (e.g. leukemia inhibitory factor and interleukin 11). OnM treatment does not affect gp130 expression levels and desensitizes signaling mediated by a transduced chimeric receptor containing extracellular domains of platelet-derived growth factor receptor-beta (PDGFRbeta) and the cytoplasmic region of gp130. Interestingly, a chimeric PDGFRbeta-gp130 mutant receptor, in which intracellular Tyr residue 759 of gp130 is replaced by a Phe residue, mediates prolonged signaling and is not cross-desensitized by OnM. Phospho-Tyr759 is the binding site for both SOCS-3 and for Src homology domain 2-containing tyrosine phosphatase 2 (SHP-2). In human aortic smooth muscle cells, neither prevention of SOCS-3 protein induction, using STAT3 or SOCS-3 antisense, nor prevention of SHP-2 expression, also with antisense, ablates desensitization. These data suggest that desensitization of vascular cells to OnM is mediated in trans and involves Tyr residue 759 in gp130 but is not mediated by either SOCS-3 or SHP-2, the only two proteins currently known to bind to gp130 at this site.

Tumor necrosis factor (TNF) activates pro-inflammatory functions of vascular endothelial cells (EC) through binding to receptor type 1 (TNFR1) molecules expressed on the cell surface. The majority of TNFR1 molecules are localized to the Golgi apparatus. Soluble forms of TNFR1 (as well as of TNFR2) can be shed from the EC surface and inhibit TNF actions. The relationships among cell surface, Golgi-associated, and shed forms of TNFR1 are unclear. Here we report that histamine causes transient loss of surface TNFR1, TNFR1 shedding, and mobilization of TNFR1 molecules from the Golgi in cultured human EC. The Golgi pool of TNFR1 serves both to replenish cell surface receptors and as a source of shed receptor. Histamine-induced shedding is blocked by TNF-alpha protease inhibitor, an inhibitor of TNF-alpha-converting enzyme, and through the H1 receptor via a MEK-1/p42 and p44 mitogen-activated protein kinase pathway. Cultured EC with histamine-induced surface receptor loss become transiently refractory to TNF. Histamine injection into human skin engrafted on immunodeficient mice similarly caused shedding of TNFR1 and diminished TNF-mediated induction of endothelial adhesion molecules. These results both clarify relationships among TNFR1 populations and reveal a novel anti-inflammatory activity of histamine.