Molecular incompatibility between pig CD200 and human CD200 receptor in in vitro xenogeneic immune responses.

Overexpression of human CD200 (hCD200) in porcine endothelial cells (PECs) has been reported to suppress xenogeneic immune responses of human macrophages against porcine endothelial cells. The current study aimed to address whether the above-mentioned beneficial effect of hCD200 is mediated by overcoming the molecular incompatibility between porcine CD200 (pCD200) and hCD200 receptor or simply by increasing the expression levels of CD200 without any molecular incompatibility across the two species. We overexpressed hCD200 or pCD200 using lentiviral vectors with V5 marker in porcine endothelial cells and compared their suppressive activity against U937-derived human macrophage-like cells (hMCs) and primary macrophages. In xenogeneic coculture of porcine endothelial cells and human macrophage-like cells or macrophages, hCD200-porcine endothelial cells suppressed phagocytosis and cytotoxicity of human macrophages to a greater extent than pCD200-porcine endothelial cells. Secretion of tumor necrosis factor-α, interleukin-1ß, and monocyte chemoattractant protein-1 from human macrophages and expression of M1 phenotypes (inducible nitric oxide synthase, dectin-1, and CD86) were also suppressed by hCD200 to a greater extent than pCD200. Furthermore, in signal transduction downstream of CD200 receptor, hCD200 induced Dok2 phosphorylation and suppressed IκB phosphorylation to a greater extent than pCD200. The above data supported the possibility of a significant molecular incompatibility between pCD200 and human CD200 receptor, suggesting that the beneficial effects of hCD200 overexpression in porcine endothelial cells could be mediated by overcoming the molecular incompatibility across the species barrier rather than by simple overexpression effects of CD200.

Anti-intercellular adhesion molecule 1 monomaintenance therapy induced long-term liver allograft survival without chronic rejection.

Calcineurin inhibitors (CNIs) are essential in liver transplantation (LT); however, their long-term use leads to various adverse effects. The anti-intercellular adhesion molecule (ICAM)-1 monoclonal antibody MD3 is a potential alternative to CNI. Despite its promising results with short-term therapy, overcoming the challenge of chronic rejection remains important. Thus, we aimed to investigate the outcomes of long-term MD3 therapy with monthly MD3 monomaintenance in nonhuman primate LT models. Rhesus macaques underwent major histocompatibility complex-mismatched allogeneic LT. The conventional immunosuppression group (Con-IS, n = 4) received steroid, tacrolimus, and sirolimus by 4 months posttransplantation. The induction MD3 group (IN-MD3, n = 5) received short-term MD3 therapy for 3 months with Con-IS. The maintenance MD3 group (MA-MD3, n = 4) received MD3 for 3 months, monthly doses by 2 years, and then quarterly. The MA-MD3 group exhibited stable liver function without overt infection and had significantly better liver allograft survival than the IN-MD3 group. Development of donor-specific antibody and chronic rejection were suppressed in the MA-MD3 group but not in the IN-MD3 group. Donor-specific T cell responses were attenuated in the MA-MD3 group. In conclusion, MD3 monomaintenance therapy without maintenance CNI provides long-term liver allograft survival by suppressing chronic rejection, offering a potential breakthrough for future human trials.

Interleukin-2/anti-interleukin-2 immune complex attenuates cold ischemia-reperfusion injury after kidney transplantation by increasing renal regulatory T cells.

BACKGROUND: Cold ischemia-reperfusion injury (IRI) is an unavoidable complication of kidney transplantation. We investigated the role of regulatory T cells (Treg) in cold IRI and whether the interleukin (IL)-2/anti-IL-2 antibody complex (IL-2C) can ameliorate cold IRI. METHODS: We developed a cold IRI mouse model using kidney transplantation and analyzed the IL-2C impact on cold IRI in acute, subacute and chronic phases. RESULTS: Treg transfer attenuated cold IRI, while Treg depletion aggravated cold IRI. Next, IL-2C administration prior to IRI mitigated acute renal function decline, renal tissue damage and apoptosis and inhibited infiltration of effector cells into kidneys and pro-inflammatory cytokine expression on day 1 after IRI. On day 7 after IRI, IL-2C promoted renal regeneration and reduced subacute renal damage. Furthermore, on day 28 following IRI, IL-2C inhibited chronic fibrosis. IL-2C decreased reactive oxygen species-mediated injury and improved antioxidant function. When IL-2C was administered following IRI, it also increased renal regeneration with Treg infiltration and suppressed renal fibrosis. In contrast, Treg depletion in the presence of IL-2C eliminated the positive effects of IL-2C on IRI. CONCLUSION: Tregs protect kidneys from cold IRI and IL-2C inhibited cold IRI by increasing the renal Tregs, suggesting a potential of IL-2C in treating cold IRI. KEY POINTS: Interleukin (IL)-2/anti-IL-2 antibody complex attenuated acute renal injury, facilitated subacute renal regeneration and suppressed chronic renal fibrosis after cold ischemia-reperfusion injury (IRI) by increasing the renal Tregs. IL-2/anti-IL-2 antibody complex decreased reactive oxygen species-mediated injury and improved antioxidant function. This study suggests the therapeutic potential of the IL-2/anti-IL-2 antibody complex in kidney transplantation-associated cold IR.

Application of microphysiologic system to assess neutrophil extracellular trap in xenotransplantation.

Transplantation of organs, cells, or tissues from one species to another, known as xenotransplantation, has the potential to alleviate organ donor shortages and enhance the success of organ transplantation. However, the possibility of immunological rejection by the recipient is one of the biggest difficulties associated with xenotransplantation. The creation of neutrophil extracellular traps (NETs), also known as NETosis, is hypothesized as a mechanism of rejection. Innovations in microfluidics and co-culturing techniques have provided access to several classes of microengineered model systems in experimental models, connecting animal research and traditional in vitro methods such as organoids, microphysiological systems, and organs-on-chip. To achieve this goal, we established a perfusable 3D Xeno vessel chip using a porcine aortic endothelial cell line and examined how NETs grow when isolated human and primate neutrophils were used. Neutrophils from both humans and monkeys displayed the usual NETosis phases, including nuclear decondensation, enlargement, and rounding of DNA, occupying the entire cytoplasm, and discharge of fragmented DNA after cell membrane rupture. Using confocal fluorescence imaging of DNA and citrullinated histone colocalization and DNA histone complex formation in supernatants from xeno vessel chips, we confirmed NETs generation by human and monkey neutrophils when cocultured in a xeno-vessel chip.

Tissue-engineered vascular microphysiological platform to study immune modulation of xenograft rejection.

Most of the vascular platforms currently being studied are lab-on-a-chip types that mimic capillary networks and are applied for vascular response analysis in vitro. However, these platforms have a limitation in clearly assessing the physiological phenomena of native blood vessels compared to in vivo evaluation. Here, we developed a simply fabricable tissue-engineered vascular microphysiological platform (TEVMP) with a three-dimensional (3D) vascular structure similar to an artery that can be applied for ex vivo and in vivo evaluation. Furthermore, we applied the TEVMP as ex vivo and in vivo screening systems to evaluate the effect of human CD200 (hCD200) overexpression in porcine endothelial cells (PECs) on vascular xenogeneic immune responses. These screening systems, in contrast to 2D in vitro and cellular xenotransplantation in vivo models, clearly demonstrated that hCD200 overexpression effectively suppressed vascular xenograft rejection. The TEVMP has a high potential as a platform to assess various vascular-related responses.

Suppressive effects of vitamin C-treated induced-regulatory T cells on heart allograft rejection under vitamin C-deficient or -sufficient conditions.

Foxp3 stability of vitamin C-treated induced-regulatory T cells (V-iTregs) is superior to that of conventional iTregs (C-iTregs). However, the role of V-iTregs in allograft rejection under vitamin C-deficient conditions, such as those seen in humans, remains unclear. We aimed to elucidate the role of vitamin C treatment on generation and maintenance of iTregs from gulo knockout (Gulo-KO) mice as well as wild type (WT) mice, and in vitro and in vivo suppressive effects of V-iTregs on heart allograft rejection in either Gulo-KO or WT recipient mice. Conversion efficiency of iTregs was similar between C- and V-iTregs in both WT and Gulo-KO mice. V-iTregs from WT or Gulo-KO mice showed better in vitro Foxp3 stability than C-iTregs, although there was no difference between WT V-iTregs and Gulo-KO V-iTregs. Furthermore, V-iTregs from WT or Gulo-KO mice suppressed in vitro T cell proliferation better than C-iTregs. Heterotrophic heart transplantation from BALB/c mice to WT or vitamin C-deficient Gulo-KO C57BL/6J mice was performed following adoptive transfer of C- or V-iTregs. V-iTregs as well as C-iTregs prolonged heart allograft survival in WT and Gulo-KO mice. However, there was no difference between the C- and V-iTreg groups. Supplementation of low- or high-dose vitamin C did not induce significant changes in heart allograft survival in Gulo-KO recipients that had received V-iTregs. In conclusion, V-iTregs do not exert better suppressive effects on heart allograft survival than C-iTregs in either WT or vitamin C-deficient recipients.

Granulocyte Colony-Stimulating Factor Attenuates Renal Ischemia-Reperfusion Injury by Inducing Myeloid-Derived Suppressor Cells.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) can increase populations of myeloid-derived suppressor cells, innate immune suppressors that play an immunoregulatory role in antitumor immunity. However, the roles of myeloid-derived suppressor cells and G-CSF in renal ischemia-reperfusion injury remain unclear. METHODS: We used mouse models of ischemia-reperfusion injury to investigate whether G-CSF can attenuate renal injury by increasing infiltration of myeloid-derived suppressor cells into kidney tissue. RESULTS: G-CSF treatment before ischemia-reperfusion injury subsequently attenuated acute renal dysfunction, tissue injury, and tubular apoptosis. Additionally, G-CSF treatment suppressed renal infiltration of macrophages and T cells as well as renal levels of IL-6, MCP-1, IL-12, TNF-α, and IFN-γ, but it increased levels of IL-10, arginase-1, and reactive oxygen species. Moreover, administering G-CSF after ischemia-reperfusion injury improved the recovery of renal function and attenuated renal fibrosis on day 28. G-CSF treatment increased renal infiltration of myeloid-derived suppressor cells (F4/80-CD11b+Gr-1int), especially the granulocytic myeloid-derived suppressor cell population (CD11b+Ly6GintLy6Clow); splenic F4/80-CD11b+Gr-1+ cells sorted from G-CSF-treated mice displayed higher levels of arginase-1, IL-10, and reactive oxygen species relative to those from control mice. Furthermore, these splenic cells effectively suppressed in vitro T cell activation mainly through arginase-1 and reactive oxygen species, and their adoptive transfer attenuated renal injury. Combined treatment with anti-Gr-1 and G-CSF showed better renoprotective effects than G-CSF alone, whereas preferential depletion of myeloid-derived suppressor cells by pep-G3 or gemcitabine abrogated the beneficial effects of G-CSF against renal injury. CONCLUSIONS: G-CSF induced renal myeloid-derived suppressor cells, thereby attenuating acute renal injury and chronic renal fibrosis after ischemia-reperfusion injury. These results suggest therapeutic potential of myeloid-derived suppressor cells and G-CSF in renal ischemia-reperfusion injury.

Human B1 Cells are the Main Blood Group A-Specific B Cells That Have a Moderate Correlation With Anti-A Antibody Titer.

BACKGROUND: Anti-carbohydrate antibody responses, including those of anti-blood group ABO antibodies, are yet to be thoroughly studied in humans. Because anti-ABO antibody-mediated rejection is a key hurdle in ABO-incompatible transplantation, it is important to understand the cellular mechanism of anti-ABO responses. We aimed to identify the main human B cell subsets that produce anti-ABO antibodies by analyzing the correlation between B cell subsets and anti-ABO antibody titers. METHODS: Blood group A-binding B cells were analyzed in peritoneal fluid and peripheral blood samples from 43 patients undergoing peritoneal dialysis and 18 healthy volunteers with blood group B or O. The correlation between each blood group A-specific B cell subset and anti-A antibody titer was then analyzed using Pearson's correlation analysis. RESULTS: Blood group A-binding B cells were enriched in CD27+CD43+CD1c- B1, CD5+ B1, CD11b+ B1, and CD27+CD43+CD1c+ marginal zone-B1 cells in peripheral blood. Blood group A-specific B1 cells (P=0.029 and R=0.356 for IgM; P=0.049 and R=0.325 for IgG) and marginal zone-B1 cells (P=0.011 and R=0.410 for IgM) were positively correlated with anti-A antibody titer. Further analysis of peritoneal B cells confirmed B1 cell enrichment in the peritoneal cavity but showed no difference in blood group A-specific B1 cell enrichment between the peritoneal cavity and peripheral blood. CONCLUSIONS: Human B1 cells are the key blood group A-specific B cells that have a moderate correlation with anti-A antibody titer and therefore constitute a potential therapeutic target for successful ABO-incompatible transplantation.

Anti-CD45RB Antibody Therapy Attenuates Renal Ischemia-Reperfusion Injury by Inducing Regulatory B Cells.

BACKGROUND: Regulatory B cells are a newly discovered B cell subset that suppresses immune responses. Recent studies found that both anti-CD45RB and anti-Tim-1 treatments regulate immune responses by inducing regulatory B cells; however, the role of these cells in renal ischemia-reperfusion injury (IRI) is unknown. METHODS: Using mouse models, including T cell-deficient (RAG1 knockout and TCRα knockout) mice and B cell-deficient (µMT) mice, we investigated the effects of regulatory B cells and anti-CD45RB on IRI and the mechanisms underlying these effects. RESULTS: Adoptive transfer of regulatory B cells before or after IRI attenuated renal IRI. Anti-CD45RB treatment with or without anti-Tim-1 before IRI increased renal infiltration of CD19+Tim-1+ regulatory B and regulatory T cells. Anti-CD45RB decreased serum creatinine levels, pathologic injury score, tubular apoptosis, and proinflammatory cytokines levels, whereas IL-10 levels increased. Following IRI, anti-CD45RB with or without anti-Tim-1 also induced regulatory B cells, improving renal function and tubular regeneration. In RAG1 knockout mice with B cell transfer, TCRα knockout mice, and wild-type mice with T cell depletion, anti-CD45RB increased regulatory B cells and attenuated IRI. However, anti-CD45RB did not attenuate IRI in RAG1 knockout mice with T cell transfer or µMT mice and induced only mild improvement in wild-type mice with B cell depletion. Furthermore, B cell-deficient mice receiving B cells from IL-10 knockout mice (but not from wild-type mice) did not show renal protection against IRI when treated with anti-CD45RB. CONCLUSIONS: Anti-CD45RB treatment attenuated acute renal injury and facilitated renal recovery after IRI through induction of IL-10+ regulatory B cells, pointing to anti-CD45RB as a potential therapeutic strategy in renal IRI.

Identification of Human B-1 Helper T Cells With a Th1-Like Memory Phenotype and High Integrin CD49d Expression.

Human B-1 cells have been proposed to be CD20+CD27+CD43+CD1c- B cells found in the umbilical cord and adult peripheral blood, but their regulatory mechanisms have not been well elucidated. Previously, we reported that mouse CD49dhigh CD4+ T cells could enhance the secretion of natural antibodies by B-1 cells. In this study, we aimed to investigate the presence and helper functions of the human equivalents of murine CD49dhigh CD4+ T cells. Here, we showed that human CD49dhigh CD4+ T cells found in the peritoneal cavity (PEC), spleen, and peripheral blood can enhance the production of IgM antibodies by B-1 cells. As revealed in mouse, CD49dhigh CD4+ T cells were more abundant in the PEC and showed a higher tendency to form conjugates with B cells than CD49dlow CD4+ T cells. Moreover, CD49dhigh CD4+ T cells showed a Th1-like memory phenotype, characterized by high expression of CD44 and CXCR3; low expression of CD62L and CCR7; rapid production of IFN-γ, tumor necrosis factor-α, and IL-2 upon stimulation with phorbol myristate acetate and ionomycin; and rapid proliferation upon stimulation with anti-CD3 and anti-CD28 antibodies. These cells also expressed high levels of PD-1, ICOS, and CD5, suggesting that they are undergoing chronic stimulation. Remarkably, CD49dhigh CD4+ T cells specifically helped B-1 cells, but not follicular memory B cells (CD27+ CD43-CD1c-) or marginal zone B cells (CD27+CD43-CD1c+), produce IgM and IgG antibodies. In parallel, the titer of human anti-blood group A IgM was positively correlated with the frequency of CD49dhigh CD4+ T cells. In conclusion, we identified human CD49dhigh CD4+ T cells with a Th1-like memory phenotype that secrete Th1 proinflammatory cytokines and help B-1 cells secrete antibodies, thereby aiding in primary defense. We suggest that these CD49dhigh CD4+ T cells are a unique type of B-cell helper T cells distinct from follicular helper T cells.

Role of Human CD200 Overexpression in Pig-to-Human Xenogeneic Immune Response Compared With Human CD47 Overexpression.

BACKGROUND: Macrophages play important roles in xenograft rejection. Here, we investigated whether overexpression of human CD200 or CD47 in porcine endothelial cells (PEC) can suppress macrophages activation in xenogeneic immune responses. METHODS: PECs and human macrophages were incubated together, harvested, and analyzed for in vitro macrophage phagocytic and cytotoxicity activity, and cytokine release. Next, PECs were injected into renal subcapsular space of humanized mice. On day 10 posttransplantation, we analyzed xenograft survival and perigraft inflammatory cell infiltrations in PEC-to-humanized mouse transplantation. RESULTS: PECs highly expressing human CD200, CD47, or both CD47/CD200 were established by lentiviral vector transduction. Both CD200 and CD47 suppressed in vitro macrophage phagocytic and cytotoxic activity against PECs; decreased TNF-α, IL-1ß, and IL-6 secretion; and increased IL-10 secretion. However, simultaneous overexpression of CD200 and CD47 did not show additive effects. Next, PECs were transplanted into NOD-scid IL-2Rg null mice, and human monocytes and lymphocytes were adoptively transferred 1 day after xenotransplantation. PEC xenograft cell death and apoptosis were decreased in the CD200-PEC and CD47/CD200-PEC groups. Perigraft infiltration of human T cells was suppressed by CD47; CD200 suppressed infiltration of human macrophages to a greater extent than CD47; and the CD47/CD200-PEC group exhibited the lowest level of leukocyte infiltration. In summary, overexpression of CD200 in PECs suppressed xenogeneic activation of human macrophages and improved survival of PEC xenografts in humanized mice; however, coexpression of CD200 and CD47 did not show additive effects. CONCLUSIONS: Therefore, overexpression of human CD200 in donor pigs could constitute a promising strategy for overcoming xenograft rejection.

VDJ gene usage among B-cell receptors in ABO-incompatible kidney transplantation determined by RNA-seq Transcriptomic analysis.

BACKGROUND: Studies on B-cell subtypes and V(D)J gene usage of B-cell receptors in kidney transplants are scarce. This study aimed to investigate V(D)J gene segment usage in ABO-incompatible (ABOi) kidney transplant (KT) patients compared to that in ABO-compatible (ABOc) KT patients. METHODS: We selected 16 ABOi KT patients with accommodation (ABOiA), 6 ABOc stable KT patients (ABOcS), and 6 ABOi KT patients with biopsy-proven acute antibody-mediated rejection (ABOiR) at day 10, whose graft tissue samples had been stored in the biorepository between 2010 and 2014. Complete transcriptomes of graft tissues were sequenced and analyzed through RNA sequencing (RNA-seq). The international ImMunoGeneTics information system (IMGT®) was used for in-depth comparison of V(D)J gene segment usage. RESULTS: The mean age of the 28 KT recipients was 43.3 ± 12.8 years, and 53.6% were male. By family, IGHV3, IGHJ4, IGLV2, and IGLJ3 gene segments were most frequently used in all groups, and their usage was not statistically different among the three patient groups. While IGKV3 was most frequently used in both the ABOiA and ABOiR groups, IGKV1 was most commonly used in the ABOcS group. In addition, while IGKJ1 was most commonly used in the ABOiA and ABOcS groups, IGKJ4 was most frequently used in the ABOiR group. According to individual gene segments, IGHV4-34 and IGHV4-30-2 were more commonly used in the ABOiR group than in the ABOiA group, and IGHV6-1 was more commonly used in the ABOcS group than in the ABOiR group. IGLV7-43 was more commonly used in the ABOcS group than in the ABOi group. However, technical variability, small sample size, and potential confounding effects of Rituximab or HLA mismatching are limitations of our study. CONCLUSIONS: Our findings suggest that RNA-seq transcriptomic analyses can provide information on the V(D)J gene usage of B-cell receptors and the mechanisms of accommodation and immune reaction in ABOi KT.

Mycobacterial cord factor enhances migration of neutrophil-like HL-60 cells by prolonging AKT phosphorylation.

Trehalose 6,6'-dimycolate (TDM), or cord factor, is a crucial stimulus of immune responses during Mycobacterium tuberculosis infection. Although TDM has immuno-stimulatory properties, including adjuvant activity and the ability to induce granuloma formation, the mechanisms underlying these remain unknown. We hypothesized that TDM stimulates transendothelial migration of neutrophils, which are the first immune cells to infiltrate the tissue upon infection. In this study, it was shown that TDM enhances N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis and transendothelial movement by prolonging AKT phosphorylation in human neutrophils. TDM induced expression of macrophage-inducible C-type lectin, a receptor for TDM, and induced secretion of pro-inflammatory cytokines and chemokines in differentiated HL-60 cells. In 2- and 3-D neutrophil migration assays, TDM-stimulated neutrophils showed increased fMLP-induced chemotaxis and transendothelial migration. Interestingly, following fMLP stimulation of TDM-activated neutrophils, AKT, a crucial kinase for neutrophil polarization and chemotaxis, showed prolonged phosphorylation at serine 473. Taken together, these data suggest that TDM modulates transendothelial migration of neutrophils upon mycobacterial infection through prolonged AKT phosphorylation. AKT may therefore be a promising therapeutic target for enhancing immune responses to mycobacterial infection.

Macrophage C-type lectin is essential for phagosome maturation and acidification during Escherichia coli-induced peritonitis.

Sepsis is a life-threatening condition caused by an uncontrolled response to bacterial infection. Impaired bactericidal activity in the host is directly associated with severe sepsis; however, the underlying regulatory mechanism(s) is largely unknown. Here, we show that MCL (macrophage C-type lectin) plays a crucial role in killing bacteria during Escherichia coli-induced peritonitis. MCL-deficient mice with E. coli-induced sepsis showed lower survival rates and reduced bacterial clearance when compared with control mice, despite similar levels of proinflammatory cytokine production. Although the ability of macrophages from MCL-deficient mice to kill bacteria was impaired, they showed normal phagocytic activity and production of reactive oxygen species. In addition, MCL-deficient macrophages showed defective phagosome maturation and phagosomal acidification after E. coli infection. Taken together, these results indicate that MCL plays an important role in host defense against E. coli infection by promoting phagosome maturation and acidification, thereby providing new insight into the role of MCL during pathogenesis of sepsis and offering new therapeutic options.

The P2X7 receptor antagonist, oxidized adenosine triphosphate, ameliorates renal ischemia-reperfusion injury by expansion of regulatory T cells.

Extracellular adenosine triphosphate (ATP) binds to purinergic receptors and, as a danger molecule, promotes inflammatory responses. Here we tested whether periodate-oxidized ATP (oATP), a P2X7 receptor (P2X7R) antagonist can attenuate renal ischemia-reperfusion injury and clarify the related cellular mechanisms. Treatment with oATP prior to ischemia-reperfusion injury decreased blood urea nitrogen, serum creatinine, the tubular injury score, and tubular epithelial cell apoptosis after injury. The infiltration of dendritic cells, neutrophils, macrophages, CD69+CD4+, and CD44+CD4+ T cells was attenuated, but renal Foxp3+CD4+ Treg infiltration was increased by oATP. The levels of IL-6 and CCL2 were reduced in the oATP group. Additionally, oATP treatment following injury improved renal function, decreased the infiltration of innate and adaptive effector cells, and increased the renal infiltration of Foxp3+CD4+ Tregs. Post-ischemia-reperfusion injury oATP treatment increased tubular cell proliferation and reduced renal fibrosis. oATP treatment attenuated renal functional deterioration after ischemia-reperfusion injury in RAG-1 knockout mice; however, Treg depletion using PC61 abrogated the beneficial effects of oATP in wild-type mice. Furthermore, oATP treatment after transfer of Tregs from wild-type mice improved the beneficial effects of Tregs on ischemia-reperfusion injury, but treatment after transfer of Tregs from P2X7R knockout mice did not. Renal ischemia-reperfusion injury was also attenuated in P2X7R knockout mice. Experiments using bone marrow chimeras established that P2X7R expression on hematopoietic cells rather than non-hematopoietic cells, such as tubular epithelial cells, plays a major role in ischemia-reperfusion injury. Thus, oATP attenuated acute renal damage and facilitated renal recovery in ischemia-reperfusion injury by expansion of Tregs.

Mincle activation enhances neutrophil migration and resistance to polymicrobial septic peritonitis.

Sepsis is a systemic inflammatory response to bacterial infection. The therapeutic options for treating sepsis are limited. Impaired neutrophil recruitment into the infection site is directly associated with severe sepsis, but the precise mechanism is unclear. Here, we show that Mincle plays a key role in neutrophil migration and resistance during polymicrobial sepsis. Mincle-deficient mice exhibited lower survival rates in experimental sepsis from cecal ligation and puncture and Escherichia coli-induced peritonitis. Mincle deficiency led to higher serum inflammatory cytokine levels and reduced bacterial clearance and neutrophil recruitment. Transcriptome analyses revealed that trehalose dimycolate, a Mincle ligand, reduced the expression of G protein-coupled receptor kinase 2 (GRK2) in neutrophils. Indeed, GRK2 expression was upregulated, but surface expression of the chemokine receptor CXCR2 was downregulated in blood neutrophils from Mincle-deficient mice with septic injury. Moreover, CXCL2-mediated adhesion, chemotactic responses, and F-actin polymerization were reduced in Mincle-deficient neutrophils. Finally, we found that fewer Mincle-deficient neutrophils infiltrated from the blood circulation into the peritoneal fluid in bacterial septic peritonitis compared with wild-type cells. Thus, our results indicate that Mincle plays an important role in neutrophil infiltration and suggest that Mincle signaling may provide a therapeutic target for treating sepsis.

IL-2/anti-IL-2 complexes ameliorate lupus nephritis by expansion of CD4+CD25+Foxp3+ regulatory T cells.

Adoptive transfer of regulatory T cells (Tregs) can delay disease progression and reduce mortality in lupus-prone mice. Here, we tested whether complex (IL-2C) consisting of IL-2 and anti-IL-2 monoclonal antibody (JES6-1) ameliorates lupus nephritis by expanding Tregs as an alternative to problematic Treg infusion therapy. IL-2C treatment of NZB/W F1 mice induced an effective and sustained expansion of CD4+CD25+Foxp3+ Tregs in both the kidneys and spleen along with decreased renal infiltration of T cells, B cells, and innate immune cells. Compared with controls, mice treated with IL-2C showed reduced proteinuria and fewer acute and chronic renal pathological lesions with improved renal function and survival. IL-2C significantly attenuated glomerular and tubular injury, vasculitis scores, and renal deposition of IgG and C3. Disease activity markers, such as high levels of anti-dsDNA antibodies and immunoglobulin levels, and low levels of complement were improved in sera of IL-2C-treated mice. IL-2C treatment decreased renal expression of TNF-α and IL-6, and the frequencies of IFN-γ+CD4+ and IL-17A+CD4+ T cells in both the kidneys and spleen. Depletion of Tregs by anti-CD25 antibodies abrogated the beneficial effects of IL-2C. When compared with combination therapy of steroid and mycophenolate mofetil, IL-2C treatment showed similar or better outcomes. Thus, IL-2C protected lupus-prone mice against lupus nephritis by expanding Tregs. Hence, IL-2C could have therapeutic potential in lupus nephritis.

Granulocyte colony-stimulating factor treatment ameliorates lupus nephritis through the expansion of regulatory T cells.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) can induce regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs). Despite the immune modulatory effects of G-CSF, results of G-CSF treatment in systemic lupus erythematosus are still controversial. We therefore investigated whether G-CSF can ameliorate lupus nephritis and studied the underlying mechanisms. METHODS: NZB/W F1 female mice were treated with G-CSF or phosphate-buffered saline for 5 consecutive days every week from 24 weeks of age, and were analyzed at 36 weeks of age. RESULTS: G-CSF treatment decreased proteinuria and serum anti-dsDNA, increased serum complement component 3 (C3), and attenuated renal tissue injury including deposition of IgG and C3. G-CSF treatment also decreased serum levels of BUN and creatinine, and ultimately decreased mortality of NZB/W F1 mice. G-CSF treatment induced expansion of CD4+CD25+Foxp3+ Tregs, with decreased renal infiltration of T cells, B cells, inflammatory granulocytes and monocytes in both kidneys and spleen. G-CSF treatment also decreased expression levels of MCP-1, IL-6, IL-2, and IL-10 in renal tissues as well as serum levels of MCP-1, IL-6, TNF-α, IL-10, and IL-17. When Tregs were depleted by PC61 treatment, G-CSF-mediated protective effects on lupus nephritis were abrogated. CONCLUSIONS: G-CSF treatment ameliorated lupus nephritis through the preferential expansion of CD4+CD25+Foxp3+ Tregs. Therefore, G-CSF has a therapeutic potential for lupus nephritis.

Protective effects of a dimeric derivative of ferulic acid in animal models of Alzheimer's disease.

Ferulic acid is a compound with potent anti-oxidant and anti-inflammatory activities. We previously reported the protective effects of ferulic acid administration against two animal models of Alzheimer's disease (AD): intracerebroventricular (i.c.v.) injection of Aß1-42 in mice and APP/PS1 mutant transgenic mice. In this study using the same AD animal models, we examined the effect of KMS4001, one of dimeric derivatives of ferulic acid. Intragastric pretreatment of mice with KMS4001 (30mg/kg/day) for 5 days significantly attenuated the Aß1-42 (i.c.v.)-induced memory impairment both in passive avoidance test and in Y-maze test. APP/PS1 mutant transgenic mice at KMS4001 doses of 3 and 30mg/kg/day via drinking water showed the significantly enhanced novel-object recognition memory at both 1.5 and 3 months after the start of KMS4001 treatment. Treatment of APP/PS1 mutant transgenic mice with KMS4001 for 3 months at the doses of 3 and 30mg/kg/day markedly decreased Aß1-40 and Aß1-42 levels in the frontal cortex. The KMS4001 dose-response relationships for Aß decrease and for improvement in novel-object recognition test corresponded to each other. Taken together, these results suggest that KMS4001 could be an effective drug candidate against AD.