IL-6 trans-signaling in a humanized mouse model of scleroderma.

Fibrosis is regulated by interactions between immune and mesenchymal cells. However, the capacity of cell types to modulate human fibrosis pathology is poorly understood due to lack of a fully humanized model system. MISTRG6 mice were engineered by homologous mouse/human gene replacement to develop an immune system like humans when engrafted with human hematopoietic stem cells (HSCs). We utilized MISTRG6 mice to model scleroderma by transplantation of healthy or scleroderma skin from a patient with pansclerotic morphea to humanized mice engrafted with unmatched allogeneic HSC. We identified that scleroderma skin grafts contained both skin and bone marrow-derived human CD4 and CD8 T cells along with human endothelial cells and pericytes. Unlike healthy skin, fibroblasts in scleroderma skin were depleted and replaced by mouse fibroblasts. Furthermore, HSC engraftment alleviated multiple signatures of fibrosis, including expression of collagen and interferon genes, and proliferation and activation of human T cells. Fibrosis improvement correlated with reduced markers of T cell activation and expression of human IL-6 by mesenchymal cells. Mechanistic studies supported a model whereby IL-6 trans-signaling driven by CD4 T cell-derived soluble IL-6 receptor complexed with fibroblast-derived IL-6 promoted excess extracellular matrix gene expression. Thus, MISTRG6 mice transplanted with scleroderma skin demonstrated multiple fibrotic responses centered around human IL-6 signaling, which was improved by the presence of healthy bone marrow-derived immune cells. Our results highlight the importance of IL-6 trans-signaling in pathogenesis of scleroderma and the ability of healthy bone marrow-derived immune cells to mitigate disease.

Complement Membrane Attack Complexes Assemble NLRP3 Inflammasomes Triggering IL-1 Activation of IFN-γ-Primed Human Endothelium.

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)-1ß expression. Inflammasome processed pro-IL-1ß, and gasdermin D results in IL-1ß 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-1ß-mediated increases in EC immunogenicity. Similar responses may underlie other complement-mediated pathologies.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium.

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-1ß, 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.

Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+ NIK+ signalosome on Rab5+ endosomes.

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.

Alloantibody and complement promote T cell-mediated cardiac allograft vasculopathy through noncanonical nuclear factor-κB signaling in endothelial cells.

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.

Reperfusion injury intensifies the adaptive human T cell alloresponse in a human-mouse chimeric artery model.

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. CONCLUSIONS: 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.

Complement-activated interferon-γ-primed human endothelium transpresents interleukin-15 to CD8+ T cells.

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-1ß and an NLRP3 inflammasome to process and secrete active IL-1ß. IL-1ß 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-1ß 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.

Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells.

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.

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes.

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.

Regulation of human T cell responses by dNP2-ctCTLA-4 inhibits human skin and microvessel graft rejection.

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-ctCTLA-4, 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.