Drew A. Roenneburg

Microchimerism is strongly correlated with tolerance to noninherited maternal antigens in mice

In mice and humans, the immunologic effects of developmental exposure to noninherited maternal antigens (NIMAs) are quite variable. This heterogeneity likely reflects differences in the relative levels of NIMA-specific T regulatory (T(R)) versus T effector (T(E)) cells. We hypothesized that maintenance of NIMA-specific T(R) cells in the adult requires continuous exposure to maternal cells and antigens (eg, maternal microchimerism [MMc]). To test this idea, we used 2 sensitive quantitative polymerase chain reaction (qPCR) tests to detect MMc in different organs of NIMA(d)-exposed H2(b) mice. MMc was detected in 100% of neonates and a majority (61%) of adults; nursing by a NIMA+ mother was essential for preserving MMc into adulthood. MMc was most prevalent in heart, lungs, liver, and blood, but was rarely detected in unfractionated lymphoid tissues. However, MMc was detectable in isolated CD4+, CD11b+, and CD11c+ cell subsets of spleen, and in lineage-positive cells in heart. Suppression of delayed type hypersensitivity (DTH) and in vivo lymphoproliferation correlated with MMc levels, suggesting a link between T(R) and maternal cell engraftment. In the absence of neonatal exposure to NIMA via breastfeeding, MMc was lost, which was accompanied by sensitization to NIMA in some offspring, indicating a role of oral exposure in maintaining a favorable T(R) > T(E) balance.

Th1 and Th17 immunocompetence in humanized NOD/SCID/IL2rγnull mice

We evaluated the immunocompetence of human T cells in humanized NOD-SCID interleukin (IL)-2r-gamma-null (hu-NSG) mice bearing a human thymic organoid, after multilineage reconstitution with isogeneic human leukocytes. Delayed type hypersensitivity (DTH) response was assessed by a direct footpad challenge of the immunized hu-NSG host, or by transfer of splenocytes from immunized hu-NSG, along with antigen, into footpads of C.B-17 scid mice (trans vivo [tv] DTH). Both methods revealed cellular immunity to tetanus toxoid (TT) or collagen type V (ColV). Immunohistochemical analysis of the swollen footpads revealed infiltration of human CD45(+) cells, including CD3(+) T cells, CD68(+) macrophages, and murine Ly6G(+) neutrophils. We observed a significant correlation between the percentage of circulating human CD4(+) cells and the direct DTH swelling response to TT. The tvDTH response to TT was inhibited by anti-interferon-gamma, whereas the tvDTH response to collagen V was inhibited by anti-IL-17 antibody, mimicking the cytokine bias of adult human T cells to these antigens. hu-NSG mice were also capable of mounting a B-cell response (primarily IgM) to TT antigen. The activation of either Th1- or Th17-dependent cellular immune response supports the utility of hu-NSG mice as a surrogate model of allograft rejection and autoimmunity.

Developmental Exposure to Noninherited Maternal Antigens Induces CD4+ T Regulatory Cells: Relevance to Mechanism of Heart Allograft Tolerance

We hypothesize that developmental exposure to noninherited maternal Ags (NIMA) results in alloantigen-specific natural and adaptive T regulatory (TR) cells. We compared offspring exposed to maternal H-2d (NIMAd) with nonexposed controls. In vitro assays did not reveal any differences in T cell responses pretransplant. Adoptive transfer assays revealed lower lymphoproliferation and greater cell surface TGF-β expression on CD4+ T cells of NIMAd-exposed vs control splenocytes. NIMAd-exposed splenocytes exhibited bystander suppression of tetanus-specific delayed-type hypersensitivity responses, which was reversed with Abs to TGF-β and IL-10. Allospecific T effector cells were induced in all mice upon i.v. challenge with B6D2F1 splenocytes or a DBA/2 heart transplant, but were controlled in NIMAd-exposed mice by TR cells to varying degrees. Some (40%) NIMAd-exposed mice accepted a DBA/2 allograft while others (60%) rejected in delayed fashion. Rejector and acceptor NIMAd-exposed mice had reduced T effector responses and increased Foxp3+ TR cells (CD4+CD25+Foxp3+ TR) in spleen and lymph nodes compared with controls. The key features distinguishing NIMAd-exposed acceptors from all other mice were: 1) higher frequency of IL-10- and TGF-β-producing cells primarily in the CD4+CD25+ T cell subset within lymph nodes and allografts, 2) a suppressed delayed-type hypersensitivity response to B6D2F1 Ags, and 3) allografts enriched in LAP+, Foxp3+, and CD4+ T cells, with few CD8+ T cells. We conclude that the beneficial NIMA effect is due to induction of NIMA-specific TR cells during ontogeny. Their persistence in the adult, and the ability of the host to mobilize them to the graft, may determine whether NIMA-specific tolerance is achieved.

Interleukin-17-dependent autoimmunity to collagen type v in atherosclerosis

Rationale: Considerable evidence shows atherosclerosis to be a chronic inflammatory disease in which immunity to self-antigens contributes to disease progression. We recently identified the collagen type V [col(V)] &agr;1(V) chain as a key autoantigen driving the Th17-dependent cellular immunity underlying another chronic inflammatory disease, obliterative bronchiolitis. Because specific induction of &agr;1(V) chains has previously been reported in human atheromas, we postulated involvement of col(V) autoimmunity in atherosclerosis. Objective: To determine whether col(V) autoimmunity may be involved in the pathogenesis of atherosclerosis. Methods and Results: Here, we demonstrate Th17-dependent anti-col(V) immunity to be characteristic of atherosclerosis in human coronary artery disease (CAD) patients and in apolipoprotein E–null (ApoE−/−) atherosclerotic mice. Responses were &agr;1(V)-specific in CAD with variable Th1 pathway involvement. In early atherosclerosis in ApoE−/− mice, anti-col(V) immunity was tempered by an interleukin (IL)-10–dependent mechanism. In support of a causal role for col(V) autoimmunity in the pathogenesis of atherosclerosis, col(V) sensitization of ApoE−/− mice on a regular chow diet overcame IL-10–mediated inhibition of col(V) autoimmunity, leading to increased atherosclerotic burden in these mice and local accumulation of IL-17–producing cells, particularly in the col(V)-rich adventitia subjacent to the atheromas. Conclusions: These findings establish col(V) as an autoantigen in human CAD and show col(V) autoimmunity to be a consistent feature in atherosclerosis in humans and mice. Furthermore, data are consistent with a causative role for col(V) in the pathogenesis of atherosclerosis.

Unaltered graft survival and intragraft lymphocytes infiltration in the cardiac allograft of Cxcr3-/- mouse recipients.

Previous studies showed that absence of chemokine receptor Cxcr3 or its blockade prolong mouse cardiac allograft survival. We evaluated the effect of the CXCR3 receptor antagonist MRL‐957 on cardiac allograft survival, and also examined the impact of anti‐CXCR3 mAb in human CXCR3 knock‐in mice. We found only a moderate increase in graft survival (10.5 and 16.6 days, p < 0.05) using either the antagonist or the antibody, respectively, compared to control (8.7 days). We re‐evaluated cardiac allograft survival with two different lines of Cxcr3−/‐ mice. Interestingly, in our hands, neither of the independently derived Cxcr3−/‐ lines showed remarkable prolongation, with mean graft survival of 9.5 and 10.8 days, respectively. There was no difference in the number of infiltrating mononuclear cells, expansion of splenic T cells or IFN‐γ production of alloreactive T cells. Mechanistically, an increased other chemokine receptor fraction in the graft infiltrating CD8 T cells in Cxcr3−/‐ recipients compared to wild‐type recipients suggested compensatory T‐cell trafficking in the absence of Cxcr3. We conclude Cxcr3 may contribute to, but does not govern, leukocyte trafficking in this transplant model.

Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways

INTRODUCTION We have previously demonstrated that transforming growth factor-β (TGF-β) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-β-induced VSMC proliferation. METHODS Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with small interfering RNA (siRNA). For in vivo experiments, male Sprague-Dawley rats underwent carotid balloon injury, followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK, and proliferating cell nuclear antigen. RESULTS In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-β-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by proliferating cell nuclear antigen. CONCLUSIONS Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-β enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

Cranberry Proanthocyanidins Improve the Gut Mucous Layer Morphology and Function in Mice Receiving Elemental Enteral Nutrition

BACKGROUND Lamina propria Th2 cytokines, interleukin (IL)-4 and IL-13, stimulate goblet cell (GC) proliferation and MUC2 production, which protect the intestinal mucosa. Elemental enteral nutrition (EEN) reduces tissue IL-4 and impairs barrier function. Proanthocyanidins (PACs) stimulate oral mucin levels. We hypothesized that adding PAC to EEN would maintain Th2-without stimulating Th1-cytokines and preserve luminal MUC2 vs EEN alone. MATERIALS AND METHODS Seventy mice were randomized to 5 diet groups-standard chow, intragastric EEN, or EEN with lowPAC, midPAC (50 mg), or highPAC (100 mg PAC/kg BW)-for 5 days, starting 2 days after gastric cannulation. Ileal tissue was analyzed for histomorphology and the cytokines IL-4, IL-13, IL-1β, IL-6, and TNF-α by enzyme-linked immunosorbent assay. MUC2 was measured in intestinal washes. RESULTS EEN lowered IL-13 (P < .05) compared with standard chow, whereas IL-4 was not significant (P < .07). LowPAC and midPAC increased IL-13 (P < .05), whereas highPAC increased both IL-4 and IL-13 (P < .05) compared with EEN. All EEN diets reduced (P < .05) crypt depth compared with the chow group. Compared with standard chow, GC numbers and luminal MUC2 were reduced with EEN (P < .05). These effects were attenuated (P < .05) with midPAC and highPAC. No changes were observed in tissue Th1 cytokines. CONCLUSIONS Adding PACs to EEN reverses impaired intestinal barrier function following EEN by improving the gut mucous layer and function through increased GC size and number as well as levels of MUC2 and ileal IL-4 and IL-13.

Human NKT cells promote monocyte differentiation into suppressive myeloid antigen-presenting cells

NKT cells have been shown to promote peripheral tolerance in a number of model systems, yet the processes by which they exert their regulatory effects remain poorly understood. Here, we show that soluble factors secreted by human NKT cells instruct human peripheral blood monocytes to differentiate into myeloid APCs that have suppressive properties. NKT‐instructed monocytes acquired a cell surface phenotype resembling myeloid DCs. However, whereas control DCs that were generated by culturing monocytes with recombinant GM‐CSF and IL‐4 had a proinflammatory phenotype characterized by the production of IL‐12 with little IL‐10, NKT‐instructed APCs showed the opposite cytokine production profile of high IL‐10 with little or no IL‐12. The control DCs efficiently stimulated peripheral blood T cell IFN‐γ secretion and proliferation, whereas NKT‐instructed APCs silenced these T cell responses. Exposure to NKT cell factors had a dominant effect on the functional properties of the DCs, since DCs differentiated by recombinant GM‐CSF and IL‐4 in the presence of NKT cell factors inhibited T cell responses. To confirm their noninflammatory effects, NKT‐instructed APCs were tested in an in vivo assay that depends on the activation of antigen‐specific human T cells. Control DCs promoted substantial tissue inflammation; however, despite a marked neutrophilic infiltrate, there was little edema in the presence of NKT‐instructed APCs, suggesting the inflammatory cascade was held in check. These results point to a novel pathway initiated by NKT cells that can contribute to the regulation of human antigen‐specific Th1 responses.

Polyelectrolyte multilayers promote stent-mediated delivery of DNA to vascular tissue.

We report an approach to deliver DNA to vascular tissue in vivo using intravascular stents coated with degradable, DNA-containing polyelectrolyte multilayers (PEMs). Ionically cross-linked multilayers ∼120 nm thick were fabricated layer-by-layer on the surfaces of balloon-mounted stainless steel stents using plasmid DNA and a hydrolytically degradable poly(β-amino ester) (polymer 1). Characterization of stents coated using a fluorescently end-labeled analog of polymer 1 revealed film erosion to be uniform across the surfaces of the stents; differential stresses experienced upon balloon expansion did not lead to faster film erosion or dose dumping of DNA in areas near stent joints when stents were incubated in physiologically relevant media. The ability of film-coated stents to transfer DNA and transfect arterial tissue in vivo was then investigated in pigs and rabbits. Stents coated with films fabricated using fluorescently labeled DNA resulted in uniform transfer of DNA to sub-endothelial tissue in the arteries of pigs in patterns corresponding to the locations and geometries of stent struts. Stents coated with films fabricated using polymer 1 and plasmid DNA encoding EGFP resulted in expression of EGFP in the medial layers of stented tissue in both pigs and rabbits two days after implantation. The results of this study, combined with the modular and versatile nature of layer-by-layer assembly, provide a polymer-based platform that is well suited for fundamental studies of stent-mediated gene transfer. With further development, this approach could also prove useful for the design of nonviral, gene-based approaches for prevention of complications that arise from the implantation of stents and other implantable interventional devices.

Analysis of the CD1 Antigen Presenting System in Humanized SCID Mice

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34+ hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a+ cells with a dendritic morphology were present in the thymic medulla. CD1+ cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens.