Toll-like Receptor 9 Induced Dendritic Cell Activation Promotes Anti-Myeloperoxidase Autoimmunity and Glomerulonephritis.

ANCA-associated vasculitis (AAV) is intricately linked with infections. Toll-like receptors (TLR) provide a potential link between infection and anti-myeloperoxidase (MPO) autoimmunity. TLR9 ligation has been shown to promote anti-MPO autoimmunity and glomerular vasculitis in murine MPO-AAV. This study investigates dendritic cell TLR9 ligation in murine experimental anti-MPO glomerulonephritis. We analyzed autoimmune responses to MPO following transfer of TLR9 stimulated, MPO pulsed dendritic cells and kidney injury following a sub-nephritogenic dose of sheep anti-mouse glomerular basement membrane globulin. TLR9 ligation enhanced dendritic cell activation upregulating CD40 and CD80 expression, promoting systemic anti-MPO autoimmunity and T cell recall responses and exacerbating kidney injury. CD40 upregulation by TLR9 was critical for the induction of nephritogenic autoimmunity. The presence of DEC205, which transports the TLR9 ligand to TLR9 located in the endosome, also promoted kidney injury. This confirms TLR9 mediated dendritic cell activation as a mechanism of anti-MPO autoimmunity in AAV and further defines the link between infection and the generation of MPO specific autoimmune inflammation.

Anti-CD20 mAb-Induced B Cell Apoptosis Generates T Cell Regulation of Experimental Myeloperoxidase ANCA-Associated Vasculitis.

BACKGROUND: Myeloperoxidase ANCA-associated vasculitis is a major cause of ESKD. Efficacy of anti-CD20 mAb treatment was tested in a mouse model of the disease. METHODS: MPO immunization induced anti-MPO autoimmunity, and a subnephritogenic dose of sheep anti-mouse GBM globulin triggered GN. RESULTS: Anti-CD20 mAb treatment increased the numbers and immunomodulatory capacity of MPO-specific T regulatory cells (Tregs) and attenuated T cell-mediated and humoral anti-MPO autoimmunity and GN. Disabling of Tregs negated the therapeutic benefit of anti-CD20 treatment. The mechanism of enhancement of Treg activity could be attributed to anti-CD20 mAb effects on inducing B cell apoptosis. Administering anti-CD20 mAb-induced apoptotic splenocytes to mice developing anti-MPO GN was as effective as anti-CD20 mAb treatment in inducing Tregs and attenuating both anti-MPO autoimmunity and GN. A nonredundant role for splenic macrophages in mediating the anti-CD20 mAb-induced immunomodulation was demonstrated by showing that administration of anti-CD20 mAb ex vivo-induced apoptotic splenocytes to unmanipulated mice attenuated autoimmunity and GN, whereas deletion of splenic marginal zone macrophages prevented anti-CD20 mAb-induced immunomodulation and treatment efficacy. Six days after administering anti-CD20 mAb to mice with murine anti-MPO GN, cell-mediated anti-MPO responses and GN were attenuated, and Tregs were enhanced, but ANCA levels were unchanged, suggesting humoral autoimmunity was redundant at this time point. CONCLUSIONS: Collectively, these data suggest that, as well as reducing humoral autoimmunity, anti-CD20 mAb more rapidly induces protective anti-MPO Treg-mediated immunomodulation by splenic processing of anti-CD20-induced apoptotic B cells.

Tolerogenic Dendritic Cells Attenuate Experimental Autoimmune Antimyeloperoxidase Glomerulonephritis.

Background Because of their capacity to induce antigen-specific immunosuppression, tolerogenic dendritic cells are a promising tool for treatment of autoimmune conditions, such as GN caused by autoimmunity against myeloperoxidase (MPO). METHODS: We sought to generate tolerogenic dendritic cells to suppress anti-MPO GN by culturing bone marrow cells with an NFκB inhibitor (BAY 11-7082) and exposing them to a pulse of MPO. After administering these MPO/BAY dendritic cells or saline to mice with established anti-MPO or anti-methylated BSA (mBSA) immunity, we assessed immune responses and GN. We also examined mechanisms of action of MPO/BAY dendritic cells. RESULTS: MPO/BAY dendritic cells decreased anti-MPO immunity and GN without inhibiting immune responses against mBSA; they also induced IL-10-producing regulatory T cells in MPO-immunized mice without affecting IL-10+ CD4+Foxp3- type 1 regulatory T cells or regulatory B cells. MPO/BAY dendritic cells did not inhibit anti-MPO immunity when CD4+Foxp3+ cells were depleted in vivo, showing that regulatory T cells are required for their effects. Coculture experiments with dendritic cells and CD4+Foxp3- or CD4+Foxp3+ cells showed that MPO/BAY dendritic cells generate Foxp3+ regulatory T cells from CD4+Foxp3- cells through several pathways, and induce IL-10+ regulatory T cells via inducible costimulator (ICOS), which was confirmed in vivo. Transfer of MPO/BAY dendritic cell-induced regulatory T cells in vivo, with or without anti-IL-10 receptor antibody, demonstrated that they suppress anti-MPO immunity and GN via IL-10. CONCLUSIONS: MPO/BAY dendritic cells attenuate established anti-MPO autoimmunity and GN in an antigen-specific manner through ICOS-dependent induction of IL-10-expressing regulatory T cells. This suggests that autoantigen-loaded tolerogenic dendritic cells may represent a novel antigen-specific therapeutic option for anti-MPO GN.

Apoptotic Cell-Induced, Antigen-Specific Immunoregulation to Treat Experimental Antimyeloperoxidase GN.

BACKGROUND: Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. METHODS: To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO- and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutrophils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. RESULTS: MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. CONCLUSIONS: These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.

Biologicals targeting T helper cell subset differentiating cytokines are effective in the treatment of murine anti-myeloperoxidase glomerulonephritis.

Anti-myeloperoxidase nephritogenic autoimmunity induces severe glomerulonephritis. To assess the therapeutic potential of monoclonal antibodies targeting T helper (Th) subset differentiation determining cytokines, we studied a murine model of anti-myeloperoxidase glomerulonephritis. The temporal participation of T helper subsets was determined by quantitating gene expression of CD4+ T-cells isolated from nephritic kidneys and cytokine production by lymphocytes from nodes draining myeloperoxidase immunization sites. Th17 cytokines (IL-17A and IL-6) rose rapidly but declined as autoimmunity matured when Th1 cytokines (IL-12 and TNF) predominated. Therefore, T helper subset participation in anti-myeloperoxidase autoimmunity is biphasic, with Th17 early and Th1 late. To confirm the functional relevance of this biphasic pattern, we compared systemic anti-myeloperoxidase autoimmunity in wild type, Th17 deficient and Th1 deficient mice. Early, Th1 deficient mice developed similar autoimmunity and glomerulonephritis to wild type mice. However, Th17 deficient mice had significantly reduced anti-myeloperoxidase autoimmunity. In late autoimmunity, Th1 deficient mice developed reduced autoimmunity and were protected from anti-myeloperoxidase glomerulonephritis. The therapeutic potential of these findings were demonstrated by neutralizing monoclonal antibodies. Targeting IL-23p19 attenuated early Th17 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not late phase disease. Targeting IL-12p35 attenuated late phase Th1 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not early autoimmunity or glomerulonephritis. Targeting both T helper subsets with an anti-IL-12p40 monoclonal antibody was effective during both early and late phases of anti-myeloperoxidase glomerulonephritis. Thus, definition of dominant T helper differentiating subsets in anti-myeloperoxidase glomerulonephritis by renal CD4+ T-cell cytokine gene expression allows effective proper phase monoclonal antibody treatment of anti-myeloperoxidase glomerulonephritis.

Natural killer cell function predicts severe infection in kidney transplant recipients.

The aim of this study was to determine if natural killer cell number (CD3- /CD16± /CD56± ) and cytotoxic killing function predicts severity and frequency of infection in kidney transplant recipients. A cohort of 168 kidney transplant recipients with stable graft function underwent assessment of natural killer cell number and functional killing capacity immediately prior to entry into this prospective study. Participants were followed for 2 years for development of severe infection, defined as hospitalization for infection. Area under receiver operating characteristic (AUROC) curves were used to evaluate the accuracy of natural killer cell number and function for predicting severe infection. Adjusted odds ratios were determined by logistic regression. Fifty-nine kidney transplant recipients (35%) developed severe infection and 7 (4%) died. Natural killer cell function was a better predictor of severe infection than natural killer cell number: AUROC 0.84 and 0.75, respectively (P = .018). Logistic regression demonstrated that after adjustment for age, transplant function, transplant duration, mycophenolate use, and increasing natural killer function (odds ratio [OR] 0.82, 95% confidence interval [CI] 0.74-0.90; P < .0001) but not natural killer number (OR 0.96, 95% CI 0.93-1.00; P = .051) remained significantly associated with a reduced likelihood of severe infection. Natural killer cell function predicts severe infection in kidney transplant recipients.

A simple score can identify kidney transplant recipients at high risk of severe infection over the following 2 years.

BACKGROUND: The aim of this study was to determine whether a composite score of simple immune biomarkers and clinical characteristics could predict severe infections in kidney transplant recipients. METHODS: We conducted a prospective study of 168 stable kidney transplant recipients who underwent measurement of lymphocyte subsets, immunoglobulins, and renal function at baseline and were followed up for 2 years for the development of any severe infections, defined as infection requiring hospitalization. A point score was developed to predict severe infection based on logistic regression analysis of factors in baseline testing. RESULTS: Fifty-nine (35%) patients developed severe infection, 36 (21%) had two or more severe infections, and 3 (2%) died of infection. A group of 19 (11%) patients had the highest predicted infectious risk (>60%), as predicted by the score. Predictive variables were mycophenolate use, graft function, CD4+, and natural killer cell number. The level of immunosuppression score had an area under the receiver operating curve of 0.75 (95% CI: 0.67-0.83). CONCLUSION: Our level of immunosuppression score for predicting the development of severe infection over 2 years has sufficient prognostic accuracy for identification of high-risk patients. This data can inform research that examines strategies to reduce the risks of infection.

Pathogenic Role for γδ T Cells in Autoimmune Anti-Myeloperoxidase Glomerulonephritis.

Myeloperoxidase (MPO) anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis results from autoimmunity to MPO. IL-17A plays a critical role in generating this form of autoimmune injury but its cell of origin is uncertain. We addressed the hypothesis that IL-17A-producing γδ T cells are a nonredundant requisite in the development of MPO autoimmunity and glomerulonephritis (GN). We studied MPO-ANCA GN in wild type, αß, or γδ T cell-deficient (C57BL/6, ßTCR-/- , and δTCR-/- respectively) mice. Both T cell populations played important roles in the generation of autoimmunity to MPO and GN. Humoral autoimmunity was dependent on intact αß T cells but was unaffected by γδ T cell deletion. Following MPO immunization, activated γδ T cells migrate to draining lymph nodes. Studies in δTCR-/- and transfer of γδ T cells to δTCR-/- mice show that γδ T cells facilitate the generation of anti-MPO autoimmunity and GN. δTCR-/- mice that received IL-17A-/- γδ T cells demonstrate that the development of anti-MPO autoimmunity and GN are dependent on γδ T cell IL-17A production. Finally, transfer of anti-MPO CD4+ T cell clones to naive δTCR-/- and wild type mice with planted glomerular MPO shows that γδ T cells are also necessary for recruitment of anti-MPO αß CD4+ effector T cells. This study demonstrates that IL-17A produced by γδ T cells plays a critical role in the pathogenesis of MPO-ANCA GN by promoting the development of MPO-specific αß T cells.

C5a receptor 1 promotes autoimmunity, neutrophil dysfunction and injury in experimental anti-myeloperoxidase glomerulonephritis.

The prospects for complement-targeted therapy in ANCA-associated vasculitis have been enhanced by a recent clinical trial in which C5a receptor 1 (C5aR1) inhibition safely replaced glucocorticoids in induction treatment. C5aR1 primes neutrophils for activation by anti-neutrophil cytoplasmic antibody (ANCA) and is therefore required in models of glomerulonephritis induced by anti-myeloperoxidase antibody. Although humoral and cellular autoimmunity play essential roles in ANCA-associated vasculitis, a role for C5aR1 in these responses has not been described. Here, we use murine models to dissect the role of C5aR1 in the generation of anti-myeloperoxidase autoimmunity and the effector responses resulting in renal injury. The genetic absence or pharmacological inhibition of C5aR1 results in reduced autoimmunity to myeloperoxidase with an attenuated Th1 response, increased Foxp3+ regulatory T cells and reduction in generation of myeloperoxidase-ANCA. These changes are mediated by C5aR1 on dendritic cells, which promotes activation, and thus myeloperoxidase autoimmunity and glomerulonephritis. We also use renal intravital microscopy to determine the effect of C5aR1 inhibition on ANCA induced neutrophil dysfunction. We found that myeloperoxidase-ANCA induce neutrophil retention and reactive oxygen species burst within glomerular capillaries. These pathological behaviors are abrogated by C5aR1 inhibition. Thus, C5aR1 inhibition ameliorates both autoimmunity and intra-renal neutrophil activation in ANCA-associated vasculitis.

Seroresponses and safety of 13-valent pneumococcal conjugate vaccination in kidney transplant recipients.

BACKGROUND: Conjugated pneumococcal vaccine is recommended for kidney transplant recipients, however, their immunogenicity and potential to trigger allograft rejection though generation of de novo anti-human leukocyte antigen antibodies has not been well studied. METHODS: Clinically stable kidney transplant recipients participated in a prospective cohort study and received a single dose of 13-valent conjugate pneumococcal vaccine. Anti-pneumococcal IgG was measured for the 13 vaccine serotypes pre and post vaccination and functional anti-pneumococcal IgG for 4 serotypes post vaccination. Anti-human leukocyte antigen antibodies antibodies were measured before and after vaccination. Kidney transplant recipients were followed clinically for 12 months for episodes of allograft rejection or invasive pneumococcal disease. RESULTS: Forty-five kidney transplant recipients participated. Median days between pre and post vaccination serology was 27 (range 21-59). Post vaccination, there was a median 1.1 to 1.7-fold increase in anti-pneumococcal IgG antibody concentrations for all 13 serotypes. Kidney transplant recipients displayed a functional antibody titer ≥1:8 for a median of 3 of the 4 serotypes. Post vaccination, there were no de novo anti-human leukocyte antigen antibodies, no episodes of biopsy proven rejection or invasive pneumococcal disease. CONCLUSION: A single dose of 13-valent conjugate pneumococcal vaccine elicits increased titers and breadth of functional anti-pneumococcal antibodies in kidney transplant recipients without stimulating rejection or donor-specific antibodies.

T cell receptor assessment in autoimmune disease requires access to the most adjacent immunologically active organ.

Next generation sequencing of T and B cell receptors is emerging as a valuable and effective method to diagnose and monitor hematopoietic malignancies. So far, this approach has not been fully explored in regard to autoimmune diseases. T cells develop in the thymus where they undergo positive and negative selection, and the autoimmune regulator (Aire) is central in the establishment of immunological tolerance. Loss of Aire leads to severe multiorgan autoimmune disease with infiltration of autoreactive T cells in affected organs. Here, we have utilized next generation sequencing technology to investigate the T cell receptor repertoire in autoimmunity induced by immunization of mice with a self-antigen, myeloperoxidase. By investigating the T cell receptor repertoire in peripheral blood, spleen and lumbar lymph nodes from naïve and immunized Aire -/- mice and wild type littermates, changes in the usage of V and J genes were evident. Our results identify TCR clonotypes which could be potential targets for immune therapy. Also, Aire -/- autoimmunity is driven by a variety of autoantigens where the autoimmune response is highly polyclonal, and access to the most adjacent immunologically active tissue is required to identify T cell receptor sequences that are potentially unique to the antigen in Aire-/- immunized mice.

Myeloperoxidase Peptide-Based Nasal Tolerance in Experimental ANCA-Associated GN.

Less toxic treatment options for patients with myeloperoxidase (MPO)-ANCA-associated GN are needed. Using an established murine model of focal necrotizing GN mediated by autoimmunity to MPO (autoimmune anti-MPO GN), we assessed the capacity for nasal tolerance induced by nasal insufflation of the immunodominant nephritogenic MPO peptide (MPO409-428) to attenuate this disease. Compared with mice that received an irrelevant immunodominant ovalbumin (OVA) peptide, OVA323-339, mice that received MPO409-428 were protected from the development of humoral and cell-mediated autoimmunity to full-length MPO and the development of GN. In mice with established anti-MPO autoimmunity, nasal insufflation of MPO409-428 as a therapeutic attenuated anti-MPO GN. To investigate the nature of this induced tolerance, we isolated CD4(+) T cells from the upper airway draining lymph nodes of both OVA323-339- and MPO409-428-tolerized mice. Adoptive transfer of CD4(+) T cells from MPO409-428- but not OVA323-339-tolerized mice to animals with established anti-MPO autoimmunity attenuated the subsequent development of GN, confirming that the immunosuppression induced by these T cells is antigen specific. Ex vivo studies showed that nasal tolerance to MPO is mediated by both conventional and induced T regulatory cells. The strong homology between the pathogenic human MPO B cell epitope recognized by ANCA in patients with acute vasculitis and the nephritogenic murine T cell MPO epitope emphasizes the clinical relevance of this study.

Mast Cell Stabilization Ameliorates Autoimmune Anti-Myeloperoxidase Glomerulonephritis.

Observations in experimental murine myeloperoxidase (MPO)-ANCA-associated vasculitis (AAV) show mast cells degranulate, thus enhancing injury as well as producing immunomodulatory IL-10. Here we report that, compared with biopsy specimens from control patients, renal biopsy specimens from 44 patients with acute AAV had more mast cells in the interstitium, which correlated with the severity of tubulointerstitial injury. Furthermore, most of the mast cells were degranulated and spindle-shaped in patients with acute AAV, indicating an activated phenotype. We hypothesized that the mast cell stabilizer disodium cromoglycate would attenuate mast cell degranulation without affecting IL-10 production. We induced anti-MPO GN by immunizing mice with MPO and a low dose of anti-glomerular basement membrane antibody. When administered before or after induction of MPO autoimmunity in these mice, disodium cromoglycate attenuated mast cell degranulation, development of autoimmunity, and development of GN, without diminishing IL-10 production. In contrast, administration of disodium cromoglycate to mast cell-deficient mice had no effect on the development of MPO autoimmunity or GN. MPO-specific CD4(+) effector T cell proliferation was enhanced by co-culture with mast cells, but in the presence of disodium cromoglycate, proliferation was inhibited and IL-10 production was enhanced. These results indicate that disodium cromoglycate blocks injurious mast cell degranulation specifically without affecting the immunomodulatory role of these cells. Thus as a therapeutic, disodium cromoglycate may substantially enhance the regulatory role of mast cells in MPO-AAV.

Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.

FcγRIIB regulates T-cell autoreactivity, ANCA production, and neutrophil activation to suppress anti-myeloperoxidase glomerulonephritis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis involves innate and adaptive immune cells in the induction of autoimmunity and in autoimmune effector responses. Most Fcγ receptors (FcγRs) activate immune cells, but FcγRIIB, found in humans and mice on B cells and innate cells, is an inhibitory receptor. Here we tested whether endogenous FcγRIIB negatively regulates autoreactivity and effector responses in experimental anti-myeloperoxidase (MPO) glomerulonephritis, using wild-type and FcγRIIB(-/-) mice. After MPO immunization, FcγRIIB(-/-) mice developed higher MPO-ANCA titers and increased anti-MPO T-cell responses. Transfer of FcγRIIB-deficient dendritic cells loaded with a nephritogenic MPO peptide (MPO409-428) into wild-type mice induced stronger autoimmunity than dendritic cells derived from wild-type mice. Transferring anti-MPO antibodies into lipopolysaccharide-primed mice resulted in increased glomerular neutrophil accumulation and injury in FcγRIIB(-/-) mice, showing a role for FcγRIIB in suppressing neutrophil activation. Inducing active autoimmunity to MPO followed by triggering T cell-mediated glomerular injury by transfer of sub-nephritogenic doses of lipopolysaccharide and anti-MPO antibodies resulted in more disease in FcγRIIB(-/-) mice. Thus, endogenous FcγRIIB negatively regulates anti-MPO autoimmunity and glomerulonephritis by dendritic cells, B cells, and neutrophils to limit MPO-ANCA production, T-cell responses, and neutrophil activation.

Absence of the lysosomal protein Limp-2 attenuates renal injury in crescentic glomerulonephritis.

In humans, mutations of the intrinsic lysosomal protein SCARB2 are associated with myoclonic epilepsy, collapsing focal and segmental glomerulosclerosis, and tubular proteinuria. Mice with deficiency of Limp-2 (the murine homologue) develop tubular proteinuria but not focal and segmental glomerulosclerosis and they have a defect in macrophage function. To further elucidate the role of Limp-2 in immune function, we induced anti-glomerular basement membrane (GBM) model of crescentic glomerulonephritis in wild-type (WT) and Limp-2(-/-) littermates by intraperitoneal injections of nephrotoxic sheep serum. Renal injury and immune responses were assessed on day 14. Compared with WT, Limp-2(-/-) mice had significantly reduced crescent formation, interstitial inflammation and a trend to reduced tubulointerstitial injury. On day 1 during the heterologous phase of the disease, albuminuria was significantly increased in WT but not in Limp-2(-/-) mice. On day 14, albuminuria and renal function were similar in the two groups. There was, however, a significant reduction in the influx of glomerular macrophages and CD4(+) T cells in Limp-2(-/-) mice. Interleukin (IL)-4 and macrophage chemoattractant protein-1 (MCP-1) mRNA expression levels were significantly reduced. Despite the reduction in numbers of infiltrating cells, flow cytometry showed no difference in macrophage or T-cell numbers in the peripheral blood from untreated mice. The systemic humoral immune response, determined by glomerular mouse immunoglobulin G (IgG) deposition and mouse anti-sheep IgG subclass production, was similar in both groups. These data suggest that absence of Limp-2 reduces inflammation in experimental crescentic glomerulonephritis with decreased macrophage and T-cell infiltration in the kidney. It suggests an important role for Limp-2 in mediating the inflammatory response.

Angiopep-2-Functionalized Lipid Cubosomes for Blood-Brain Barrier Crossing and Glioblastoma Treatment.

Glioblastoma multiforme (GBM) is an aggressive brain cancer with high malignancy and resistance to conventional treatments, resulting in a bleak prognosis. Nanoparticles offer a way to cross the blood-brain barrier (BBB) and deliver precise therapies to tumor sites with reduced side effects. In this study, we developed angiopep-2 (Ang2)-functionalized lipid cubosomes loaded with cisplatin (CDDP) and temozolomide (TMZ) for crossing the BBB and providing targeted glioblastoma therapy. Developed lipid cubosomes showed a particle size of around 300 nm and possessed an internal ordered inverse primitive cubic phase, a high conjugation efficiency of Ang2 to the particle surface, and an encapsulation efficiency of more than 70% of CDDP and TMZ. In vitro models, including BBB hCMEC/D3 cell tight monolayer, 3D BBB cell spheroid, and microfluidic BBB/GBM-on-a-chip models with cocultured BBB and glioblastoma cells, were employed to study the efficiency of the developed cubosomes to cross the BBB and showed that Ang2-functionalized cubosomes can penetrate the BBB more effectively. Furthermore, Ang2-functionalized cubosomes showed significantly higher uptake by U87 glioblastoma cells, with a 3-fold increase observed in the BBB/GBM-on-a-chip model as compared to that of the bare cubosomes. Additionally, the in vivo biodistribution showed that Ang2 modification could significantly enhance the brain accumulation of cubosomes in comparison to that of non-functionalized particles. Moreover, CDDP-loaded Ang2-functionalized cubosomes presented an enhanced toxic effect on U87 spheroids. These findings suggest that the developed Ang2-cubosomes are prospective for improved BBB crossing and enhanced delivery of therapeutics to glioblastoma and are worth pursuing further as a potential application of nanomedicine for GBM treatment.

Myeloperoxidase (MPO)-specific CD4+ T cells contribute to MPO-anti-neutrophil cytoplasmic antibody (ANCA) associated glomerulonephritis.

Autoimmunity to the neutrophil enzyme myeloperoxidase (MPO) is an important cause of rapidly progressive glomerulonephritis, but the relative roles of MPO-specific anti-neutrophil cytoplasmic antibodies (MPO-ANCA) and autoreactive effector MPO-specific CD4(+) T cells are unclear. We confirmed that passive transfer of murine MPO-ANCA to agammaglobulinemic µMT mice immunized with OVA induces glomerular injury with capillary wall thickening, fibrinoid necrosis, mesangial cell proliferation, and periglomerular cell infiltration. Preimmunization of µMT mice with MPO induced MPO-specific CD4(+) T cells and significantly enhanced renal injury after MPO-ANCA transfer. CD4(+) T cell depletion prevented this augmentation of injury, confirming the importance of effector T cells in the development of MPO-ANCA associated glomerulonephritis. Therefore, MPO-ANCA can induce glomerulonephritis through both direct humoral mechanisms (recruitment of neutrophils and deposition of MPO) and indirectly by initiating MPO deposition in glomeruli, thereby directing effector CD4(+) T cell mediated injury. To confirm and support this data, we transferred T cells from MPO-immunized Mpo(-/-)µMT mice into Rag1(-/-) mice (control mice received ovalbumin specific T cells) and triggered injury by passive MPO-ANCA. Renal injury was significantly greater in mice transferred with T cells from MPO-immunized mice. These current studies demonstrate that MPO-ANCA induces injury via both humoral and cell mediated immune mechanisms.

Mast cells contribute to peripheral tolerance and attenuate autoimmune vasculitis.

Mast cells contribute to the modulation of the immune response, but their role in autoimmune renal disease is not well understood. Here, we induced autoimmunity resulting in focal necrotizing GN by immunizing wild-type or mast cell-deficient (Kit(W-sh/W-sh)) mice with myeloperoxidase. Mast cell-deficient mice exhibited more antimyeloperoxidase CD4+ T cells, enhanced dermal delayed-type hypersensitivity responses to myeloperoxidase, and more severe focal necrotizing GN. Furthermore, the lymph nodes draining the sites of immunization had fewer Tregs and reduced production of IL-10 in mice lacking mast cells. Reconstituting these mice with mast cells significantly increased the numbers of Tregs in the lymph nodes and attenuated both autoimmunity and severity of disease. After immunization with myeloperoxidase, mast cells migrated from the skin to the lymph nodes to contact Tregs. In an ex vivo assay, mast cells enhanced Treg suppression through IL-10. Reconstitution of mast cell-deficient mice with IL-10-deficient mast cells led to enhanced autoimmunity to myeloperoxidase and greater disease severity compared with reconstitution with IL-10-intact mast cells. Taken together, these studies establish a role for mast cells in mediating peripheral tolerance to myeloperoxidase, protecting them from the development of focal necrotizing GN in ANCA-associated vasculitis.

Corrigendum: Phase I trial outcome of amnion cell therapy in patients with ischemic stroke (I-ACT).

[This corrects the article DOI: 10.3389/fnins.2023.1153231.].