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BackgroundRepeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA).MethodsWe polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria.ResultsAt transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan–Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28).ConclusionThis study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.

IntroductionImmunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N-linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares.MethodsUsing liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV.ResultsAnalysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher ΔFc-bisection at diagnosis (P = 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse (P = 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse (P = 0.02).DiscussionOur data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre.

BackgroundGiant Cell Arteritis (GCA) and Polymyalgia Rheumatica (PMR) are overlapping inflammatory diseases. Antigen-presenting cells (APCs), including monocytes and dendritic cells (DCs), are main contributors to the immunopathology of GCA and PMR. However, little is known about APC phenotypes in the peripheral blood at the time of GCA/PMR diagnosis.MethodsAPCs among peripheral blood mononuclear cells (PBMCs) of treatment-naive GCA and PMR patients were compared to those in age- and sex-matched healthy controls (HCs) using flow cytometry (n=15 in each group). We identified three monocyte subsets, and three DC subsets: plasmacytoid DCs (pDCs), CD141+ conventional DCs (cDC1) and CD1c+ conventional DCs (cDC2). Each of these subsets was analyzed for expression of pattern recognition receptors (TLR2, TLR4), immune checkpoints (CD86, PDL1, CD40) and activation markers (HLA-DR, CD11c).Resultst-SNE plots revealed a differential clustering of APCs between GCA/PMR and HCs. Further analyses showed shifts in monocyte subsets and a lower proportion of the small population of cDC1 cells in GCA/PMR, whereas cDC2 proportions correlated negatively with CRP (r=-0.52). Classical monocytes of GCA/PMR patients show reduced expression of TLR2, HLA-DR, CD11c, which was in contrast to non-classical monocytes that showed higher marker expression. Additionally, single cell RNA sequencing in GCA patients identified a number of differentially expressed genes related to inflammation and metabolism in APCs.ConclusionCirculating non-classical monocytes display an activated phenotype in GCA/PMR patients at diagnosis, whereas classical monocytes show reduced expression of activation markers. Whether these findings reflect APC migration patterns or the effects of long-term inflammation remains to be investigated.

Objectives Giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) are age-associated inflammatory diseases that frequently overlap. Both diseases require long-term treatment with glucocorticoids (GCs), often associated with comorbidities. Previous population-based cohort studies reported that an unhealthier metabolic profile might prevent the development of GCA. Here, we report metabolic features before start of treatment and during treatment in patients with GCA and PMR.Methods In the Dutch GCA/PMR/SENEX (GPS) cohort, we analysed metabolic features and prevalence of comorbidities (type 2 diabetes, hypercholesterolaemia, hypertension, obesity and cataract) in treatment-naïve patients with GCA (n=50) and PMR (n=42), and compared those with the population-based Lifelines cohort (n=91). To compare our findings in the GPS cohort, we included data from patients with GCA (n=52) and PMR (n=25) from the Aarhus cohort. Laboratory measurements, comorbidities and GC use were recorded for up to 5 years in the GPS cohort.Results Glycated haemoglobin levels tended to be higher in treatment-naïve patients with GCA, whereas high-density lipoprotein, low-density lipoprotein and cholesterol levels were lower compared with the Lifelines population. Data from the Aarhus cohort were aligned with the findings obtained in the GPS cohort. Presence of comorbidities at baseline did not predict long-term GC requirement. The incidence of diabetes, obesity and cataract among patients with GCA increased upon initiation of GC treatment.Conclusion Data from the GCA and PMR cohorts imply a metabolic dysregulation in treatment-naïve patients with GCA, but not in patients with PMR. Treatment with GCs led to the rise of comorbidities and an unhealthier metabolic profile, stressing the need for prednisone-sparing targeted treatment in these vulnerable patients.

Abstract Background Diagnosing patients with giant cell arteritis (GCA) remains difficult. Due to its non-specific symptoms, it is challenging to identify GCA in patients presenting with symptoms of polymyalgia rheumatica (PMR), which is a more common disease. Also, commonly used acute-phase markers CRP and ESR fail to discriminate GCA patients from PMR and (infectious) mimicry patients. Therefore, we investigated biomarkers reflecting vessel wall inflammation for their utility in the accurate diagnosis of GCA in two international cohorts. Methods Treatment-naïve GCA patients participated in the Aarhus AGP cohort (N = 52) and the Groningen GPS cohort (N = 48). The AGP and GPS biomarker levels and symptoms were compared to patients presenting phenotypically as isolated PMR, infectious mimicry controls and healthy controls (HCs). Serum/plasma levels of 12 biomarkers were measured by ELISA or Luminex. Results In both the AGP and the GPS cohort, we found that weight loss, elevated erythrocyte sedimentation rate (ESR) and higher angiopoietin-2/-1 ratios but lower matrix metalloproteinase (MMP)-3 levels identify concomitant GCA in PMR patients. In addition, we confirmed that elevated platelet counts are characteristic of GCA but not of GCA mimicry controls and that low MMP-3 and proteinase 3 (PR3) levels may help to discriminate GCA from infections. Conclusion This study, performed in two independent international cohorts, consistently shows the potential of angiopoietin-2/-1 ratios and MMP-3 levels to identify GCA in patients presenting with PMR. These biomarkers may be used to select which PMR patients require further diagnostic workup. Platelet counts may be used to discriminate GCA from GCA look-alike patients.

ABSTRACT Porphyromonas gingivalis is a keystone oral pathogen that successfully manipulates the human innate immune defenses, resulting in a chronic proinflammatory state of periodontal tissues and beyond. Here, we demonstrate that secreted outer membrane vesicles (OMVs) are deployed by P. gingivalis to selectively coat and activate human neutrophils, thereby provoking degranulation without neutrophil killing. Secreted granule components with antibacterial activity, especially LL-37 and myeloperoxidase (MPO), are subsequently degraded by potent OMV-bound proteases known as gingipains, thereby ensuring bacterial survival. In contrast to neutrophils, the P. gingivalis OMVs are efficiently internalized by macrophages and epithelial cells. Importantly, we show that neutrophil coating is a conserved feature displayed by OMVs of at least one other oral pathogen, namely, Aggregatibacter actinomycetemcomitans. We conclude that P. gingivalis deploys its OMVs for a neutrophil-deceptive strategy to create a favorable inflammatory niche and escape killing. IMPORTANCE Severe periodontitis is a dysbiotic inflammatory disease that affects about 15% of the adult population, making it one of the most prevalent diseases worldwide. Importantly, periodontitis has been associated with the development of nonoral diseases, such as rheumatoid arthritis, pancreatic cancer, and Alzheimer’s disease. Periodontal pathogens implicated in periodontitis can survive in the oral cavity only by avoiding the insults of neutrophils while at the same time promoting an inflamed environment where they successfully thrive. Our present findings show that outer membrane vesicles secreted by the keystone pathogen Porphyromonas gingivalis provide an effective delivery tool of virulence factors that protect the bacterium from being killed while simultaneously activating human neutrophils.

BackgroundAlthough polymyalgia rheumatica (PMR) is a very common rheumatic inflammatory disease, current insight into the pathobiology of PMR is limited and largely based on studies in blood. We investigated T helper 1 (TH1) and T helper 17 (TH17) cell responses in blood, synovial fluid and bursa tissue of patients with PMR.Materials and methodsBlood samples were collected from 18 patients with new-onset PMR and 32 healthy controls. Synovial fluid was aspirated from the inflamed shoulder bursae or biceps tendon sheath of 13 patients. Ultrasound-guided biopsies of the subacromial-subdeltoid (SASD) bursa were obtained from 11 patients. T cells were examined by flow cytometry, immunohistochemistry and immunofluorescence staining.ResultsBesides an increase of TH17 (CD4+IL-17+IFN-γ-) cells and T cytotoxic 17 (TC17; CD8+IL-17+IFN-γ-) cells, no other major changes were noted in the circulating T cell compartment of patients with PMR. Absolute numbers of CD4+ and CD8+ T cells were similar in blood and synovial fluid of patients with PMR. Synovial fluid T cells showed an effector-memory (CD45RO+CCR7-) phenotype. Percentages of TH1 (CD4+IFN-γ+IL-17-) cells and TH1/TH17 (CD4+IFN-γ+IL-17+) cells, but not TH17 or TC17 cells, were increased in the synovial fluid. Bursa tissue biopsies contained a small number of T cells, which were mostly CD8 negative. The majority of bursa tissue T cells produced IFN-γ but not IL-17. For comparison, B cells were scarcely detected in the bursa tissue.ConclusionAlthough the circulating TH17 cell pool is expanded in patients with PMR, our findings indicate that TH1 cells are involved in the inflammation of bursae and tendon sheaths in this condition. Our study points towards the TH1 cell pathway as a potential target for therapy in PMR.

Giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) are two interrelated inflammatory diseases affecting patients above 50 years of age. Patients with GCA suffer from granulomatous inflammation of medium- to large-sized arteries. This inflammation can lead to severe ischemic complications (e.g., irreversible vision loss and stroke) and aneurysm-related complications (such as aortic dissection). On the other hand, patients suffering from PMR present with proximal stiffness and pain due to inflammation of the shoulder and pelvic girdles. PMR is observed in 40–60% of patients with GCA, while up to 21% of patients suffering from PMR are also affected by GCA. Due to the risk of ischemic complications, GCA has to be promptly treated upon clinical suspicion. The treatment of both GCA and PMR still heavily relies on glucocorticoids (GCs), although novel targeted therapies are emerging. Imaging has a central position in the diagnosis of GCA and PMR. While [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) has proven to be a valuable tool for diagnosis of GCA and PMR, it possesses major drawbacks such as unspecific uptake in cells with high glucose metabolism, high background activity in several non-target organs and a decrease of diagnostic accuracy already after a short course of GC treatment. In recent years, our understanding of the immunopathogenesis of GCA and, to some extent, PMR has advanced. In this review, we summarize the current knowledge on the cellular heterogeneity in the immunopathology of GCA/PMR and discuss how recent advances in specific tissue infiltrating leukocyte and stromal cell profiles may be exploited as a source of novel targets for imaging. Finally, we discuss prospective novel PET radiotracers that may be useful for the diagnosis and treatment monitoring in GCA and PMR.

Clinical observations have shown that obesity is associated with the severe outcome of SARS-CoV-2 infection hallmarked by microvascular dysfunction in the lungs and other organs. Excess visceral fat and high systemic levels of adipose tissue (AT) derived mediators such as leptin and other adipokines have also been linked to endothelial dysfunction. Consequently, we hypothesized that AT-derived mediators may exacerbate microvascular dysfunction during of SARS-CoV-2 infection and tested this in a primary human lung microvascular endothelial (HLMVEC) cell model. Our results indicate that HLMVEC are not susceptible to SARS-CoV-2 infection since no expression of viral proteins and no newly produced virus was detected. In addition, exposure to the virus did not induce endothelial activation as evidenced by a lack of adhesion molecule, E-selectin, VCAM-1, ICAM-1, and inflammatory cytokine IL-6 induction. Incubation of endothelial cells with the pro-inflammatory AT-derived mediator, leptin, prior to virus inoculation, did not alter the expression of endothelial SARS-CoV-2 entry receptors and did not alter their susceptibility to infection. Furthermore, it did not induce inflammatory activation of endothelial cells. To verify if the lack of activated phenotype in the presence of adipokines was not leptin-specific, we exposed endothelial cells to plasma obtained from critically ill obese COVID-19 patients. Plasma exposure did not result in E-selectin, VCAM-1, ICAM-1, or IL-6 induction. Together our results strongly suggest that aberrant inflammatory endothelial responses are not mounted by direct SARS-CoV-2 infection of endothelial cells, even in the presence of leptin and other mediators of obesity. Instead, endothelial activation associated with COVID-19 is likely a result of inflammatory responses initiated by other cells. Further studies are required to investigate the mechanisms regulating endothelial behavior in COVID-19 and the mechanisms driving severe disease in obese individuals.

Abstract Background Immune checkpoints are crucial molecules in maintaining a proper immune balance. Even though age and sex are known to have effects on the immune system, the interplay between age, sex and immune checkpoint expression by T cells is not known. The aim of this study was to determine whether age and sex affect immune checkpoint expression by T cells and if age and sex affect the kinetics of immune checkpoint expression following ex vivo stimulation. In this study, whole blood samples of 20 healthy young adults (YA, 9 males and 11 females) and 20 healthy older adults (OA, 9 males and 11 females) were stained for lymphocyte lineage markers and immune checkpoints and frequencies of CD28+, PD-1+, VISTA+ and CD40L+ T cells were determined. Immune checkpoint expression kinetics were studied following ex vivo anti-CD3/anti-CD28 stimulation of T cells from young and older healthy adults. Results We report an age-associated increase of CD40L + CD4+ and CD40L + CD8+ T-cell frequencies, whereas CD40+ B-cell frequencies were decreased in older adults, suggesting modulation of the CD40L-CD40 interaction with age. Immune checkpoint expression kinetics revealed differences in magnitude between CD4+ and CD8+ T cells independent of age and sex. Further analysis of CD4+ T-cell subsets revealed an age-associated decrease of especially PD-1 + CD4+ memory T cells which tracked with the female sex. Conclusion Collectively, our results demonstrate that both age and sex modulate expression of immune checkpoints by human T cells. These findings may have implications for optimising vaccination and immune checkpoint immunotherapy and move the field towards precision medicine in the management of older patient groups.

BACKGROUND Little is known about the autoreactive B cells in antineutrophil cytoplasmic antibody–associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3–reactive (PR3+) B cells.METHODS Multicolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets).RESULTS The frequency of PR3+ B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%–6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%–5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%–2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3+ B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001).CONCLUSION This study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells.Trial registration ClinicalTrials.gov NCT00104299.Funding The Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.

Autoreactivity to myeloperoxidase (MPO) causes autoimmune vasculitis and severe glomerulonephritis. Here, Ooi et al. show that a Staphylococcus aureus plasmid encodes a peptide that is homologous to an immunodominant MPO epitope and induces anti-MPO autoimmunity and glomerulonephritis in mice.

Vasculitis refers to inflammation of blood vessels and can cause a variety of serious complications depending on which vessels are affected. Two different forms of vasculitis are Giant Cell Arteritis (GCA) and Granulomatosis with Polyangiitis (GPA). GCA is the most common form of vasculitis in adults affecting the large arteries and can lead to visual impairment and development of aneurysms. GPA affects small- and medium-sized blood vessels predominantly in the lungs and kidneys resulting in organ failure. Both diseases can potentially be fatal. Although the pathogenesis of GCA and GPA are incompletely understood, a prominent role for CD4+ T cells has been implicated in both diseases. More recently, the role of CD8+ T cells has gained renewed interest. CD8+ T cells are important players in the adaptive immune response against intracellular microorganisms. After a general introduction on the different forms of vasculitis and their association with infections and CD8+ T cells, we review the current knowledge on CD8+ T-cell involvement in the immunopathogenesis of GCA and GPA focusing on phenotypic and functional features of circulating and lesional CD8+ T cells. Furthermore, we discuss to which extent aging is associated with CD8+ T-cell phenotype and function in GCA and GPA.

Granulomatosis with polyangiitis (GPA) is an autoimmune disease affecting mainly small blood vessels. B-cells are important in the GPA pathogenesis as precursors of autoantibody-producing cells but likely also contribute (auto)antibody-independently. This has been underlined by the effectiveness of B-cell-depletion (with Rituximab) in inducing and maintaining disease remission. Mycophenolate-mofetil (MMF) and azathioprine (AZA) are immunosuppressive therapies frequently used in GPA-patients. Interestingly, MMF-treated GPA-patients are more prone to relapses than AZA-treated patients, while little is known about the influence of these drugs on B-cells. We investigated whether MMF or AZA treatment (or their active compounds) alters the circulating B-cell subset distribution and has differential effects on in vitro B-cell proliferation and cytokine production in GPA-patients that might underlie the different relapse rate. Circulating B-cell subset frequencies were determined in samples from AZA-treated (n = 13), MMF-treated (n = 12), untreated GPA-patients (n = 19) and matched HCs (n = 41). To determine the ex vivo effects of the active compounds of MMF and AZA, MPA and 6-MP respectively, on B-cell proliferation and cytokine production, PBMCs of untreated GPA-patients (n = 29) and matched HCs (n = 30) were cultured for 3-days in the presence of CpG-oligodeoxynucleotides (CpG) with MPA or 6-MP. After restimulation (with phorbol myristate acetate, calcium-ionophore), cytokine-positive B-cell frequencies were measured. Finally, to assess the effect of MMF or AZA treatment on in vitro B-cell proliferation and cytokine production, PBMCs of MMF-treated (n = 18), and AZA-treated patients (n = 28) and HCs (n = 41) were cultured with CpG. The memory B-cell frequency was increased in AZA- compared to MMF-treated patients, while no other subset was different. The active compounds of MMF and AZA showed in vitro that MPA decreased B-cell proliferation in GPA-patients and HCs. B-cell proliferation in MMF- and AZA-treated patients was not different. Finally, the IL-6+ B-cell frequency was decreased by MPA compared to 6-MP. No differences in IL-10+, IL-6+ or TNFα+ B-cell proportions or proliferation were found in MMF- and AZA-treated patients. Our results indicate that MMF could be superior to AZA in inhibiting B-cell cytokine production in GPA-patients. Future studies should assess the effects of these immunosuppressive drugs on other immune cells to elucidate mechanisms underlying the potential differences in relapse rates.

Abstract Objective To determine the presence and spatial distribution of different macrophage phenotypes, governed by granulocyte macrophage colony‐stimulating factor (GM‐CSF) and macrophage colony‐stimulating factor (M‐CSF) skewing signals, in giant cell arteritis (GCA) lesions. Methods Temporal artery biopsies (TABs, n = 11) from treatment‐naive GCA patients, aorta samples from GCA‐related aneurysms (n = 10) and atherosclerosis (n = 10) were stained by immunohistochemistry targeting selected macrophage phenotypic markers, cytokines, matrix metalloproteinases (MMPs) and growth factors. In vitro macrophage differentiation (n = 10) followed by flow cytometry, Luminex assay and ELISA were performed to assess whether GM‐CSF and M‐CSF are drivers of macrophage phenotypic heterogeneity. Results A distinct spatial distribution pattern of macrophage phenotypes in TABs was identified. CD206+/MMP‐9+ macrophages were located at the site of tissue destruction, whereas FRβ+ macrophages were located in the inner intima of arteries with high degrees of intimal hyperplasia. Notably, this pattern was also observed in macrophage‐rich areas in GCA aortas but not in atherosclerotic aortas. Flow cytometry showed that GM‐CSF treatment highly upregulated CD206 expression, while FRβ was expressed by M‐CSF‐skewed macrophages, only. Furthermore, localised expression of GM‐CSF and M‐CSF was detected, likely contributing to macrophage heterogeneity in the vascular wall. Conclusions Our data document a distinct spatial distribution pattern of CD206+/MMP‐9+ macrophages and FRβ+ macrophages in GCA linked to tissue destruction and intimal proliferation, respectively. We suggest that these distinct macrophage phenotypes are skewed by sequential GM‐CSF and M‐CSF signals. Our study adds to a better understanding of the development and functional role of macrophage phenotypes in the pathogenesis of GCA and opens opportunities for the design of macrophage‐targeted therapies.

Abstract Monocytes/macrophages are critical in systemic and local inflammation in giant cell arteritis (GCA) and possibly in clinically overlapping polymyalgia rheumatica (PMR). Therefore, we aimed to understand the contribution of monocyte subsets and the CX3CR1-CX3CL1 and CCR2-CCL2 migratory pathways, to the pathology of GCA. Peripheral blood monocytes were enumerated in samples from newly-diagnosed, untreated GCA and PMR patients and after prednisone-induced remission. The distribution of classical (CD14brightCD16neg) and the more pro-inflammatory, intermediate (CD14brightCD16+) and non-classical (CD14dimCD16+) monocyte subsets was analysed by flow cytometry. The phenotype of macrophages in temporal artery biopsies (TABs) from GCA patients was studied by immunohistochemistry and immunofluorescence. A clear monocytosis was seen in newly diagnosed GCA and PMR patients caused by elevated numbers of classical monocytes. Prednisone treatment suppressed numbers of non-classical monocytes. Both chemokine CX3CL1 and CCL2 were highly expressed in the TAB. Most macrophages in the TAB of GCA patients expressed non-classical monocyte markers CD16 and CX3CR1 whereas co-localisation of CD16 with classical monocyte marker CCR2 was infrequent. In conclusion, we report an altered distribution of monocyte subsets in both GCA and PMR patients. The majority of macrophages in TABs of GCA patients were CD68 + CD16 + CX3CR1 + CCR2− and thereby resembled the phenotype of non-classical monocytes.

Abstract Obesity-related albuminuria is associated with decline of kidney function and is considered a first sign of diabetic nephropathy. Suggested factors linking obesity to kidney dysfunction include low-grade inflammation, insulin resistance and adipokine dysregulation. Here, we investigated the effects of two pharmacological compounds with established anti-inflammatory properties, rosiglitazone and rosuvastatin, on kidney dysfunction during high-fat diet (HFD)-induced obesity. For this, human CRP transgenic mice were fed standard chow, a lard-based HFD, HFD+rosuvastatin or HFD+rosiglitazone for 42 weeks to study effects on insulin resistance; plasma inflammatory markers and adipokines; and renal pathology. Rosiglitazone but not rosuvastatin prevented HFD-induced albuminuria and renal fibrosis and inflammation. Also, rosiglitazone prevented HFD-induced KIM-1 expression, while levels were doubled with rosuvastatin. This was mirrored by miR-21 expression, which plays a role in fibrosis and is associated with renal dysfunction. Plasma insulin did not correlate with albuminuria. Only rosiglitazone increased circulating adiponectin concentrations. In all, HFD-induced albuminuria, and renal inflammation, injury and fibrosis is prevented by rosiglitazone but not by rosuvastatin. These beneficial effects of rosiglitazone are linked to lowered miR-21 expression but not connected with the selectively enhanced plasma adiponectin levels observed in rosiglitazone-treated animals.

Abstract Background Persistent expansion of circulating CD4+ effector memory T cells (TEM) in patients with granulomatosis with polyangiitis (GPA) suggests their fundamental role in disease pathogenesis. Recent studies have shown that distinct functional CD4+ TEM cell subsets can be identified based on expression patterns of chemokine receptors. The current study aimed to determine different CD4+ TEM cell subsets based on chemokine receptor expression in peripheral blood of GPA patients. Identification of particular circulating CD4+ TEM cells subsets may reveal distinct contributions of specific CD4+ TEM subsets to the disease pathogenesis in GPA. Method Peripheral blood of 63 GPA patients in remission and 42 age- and sex-matched healthy controls was stained immediately after blood withdrawal with fluorochrome-conjugated antibodies for cell surface markers (CD3, CD4, CD45RO) and chemokine receptors (CCR4, CCR6, CCR7, CRTh2, CXCR3) followed by flow cytometry analysis. CD4+ TEM memory cells (CD3+CD4+CD45RO+CCR7-) were gated, and the expression patterns of chemokine receptors CXCR3+CCR4-CCR6-CRTh2-, CXCR3-CCR4+CCR6-CRTh2+, CXCR3-CCR4+CCR6+CRTh2-, and CXCR3+CCR4-CCR6+CRTh2- were used to distinguish TEM1, TEM2, TEM17, and TEM17.1 cells, respectively. Results The percentage of CD4+ TEM cells was significantly increased in GPA patients in remission compared to HCs. Chemokine receptor co-expression analysis within the CD4+ TEM cell population demonstrated a significant increase in the proportion of TEM17 cells with a concomitant significant decrease in the TEM1 cells in GPA patients compared to HC. The percentage of TEM17 cells correlated negatively with TEM1 cells in GPA patients. Moreover, the circulating proportion of TEM17 cells showed a positive correlation with the number of organs involved and an association with the tendency to relapse in GPA patients. Interestingly, the aberrant distribution of TEM1 and TEM17 cells is modulated in CMV- seropositive GPA patients. Conclusions Our data demonstrates the identification of different CD4+ TEM cell subsets in peripheral blood of GPA patients based on chemokine receptor co-expression analysis. The aberrant balance between TEM1 and TEM17 cells in remission GPA patients, showed to be associated with disease pathogenesis in relation to organ involvement, and tendency to relapse.

Abstract Background Macrophages may present two distinct phenotypes indicated as M1 and M2 under different stimuli. M1 and M2 macrophages have divergent functions that range from enhancement of inflammation for M1 to tissue repair and remodeling for M2 macrophages. The objective of this study was to evaluate the distribution of M1 and M2 macrophage phenotypes in biopsies from the airways of patients with active granulomatosis with polyangiitis (GPA) and to analyze their associations with T and B cells in those biopsies, and with nasal carriage of Staphylococcus aureus, disease parameters and therapy. Methods Consecutive GPA patients (n = 35) with active airway disease, who underwent respiratory tract biopsy were included. Immunohistochemical evaluation was performed to assess the distribution of macrophages and T and B cells using the markers CD68, CD3 and CD20, respectively. CD86 was used as the M1 marker and CD163 as the M2 marker while Tbet and GATA-3 were used as Th1 and Th2 markers, respectively. At the time of the biopsy patients were assessed for nasal carriage of Staphylococcus aureus and treatment. Results Percentages of macrophages and T cells were significantly higher than those of B cells in lesional tissue from the respiratory tract in GPA. M2 macrophages and Th2 cells were more frequent than M1 macrophages (p = 0.0007) and Th1 cells (p

Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression.Methods: RNA isolated from FACS-sorted memory (M) Tregs (CD4+CD45RO+CD25+CD127−) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in MTregs from patients and HCs. cAMP levels were measured using flow cytometry.Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in MTregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of MTregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = −0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, MTregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed MTregs by forskolin restored their suppressive function in vitro.Conclusion: Overexpression of miR-142-3p in MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.

Background: Granulomatosis with polyangiitis (GPA) patients are prone to disease relapses. We aimed to determine whether GPA patients at risk for relapse can be identified by differences in B cell subset frequencies.Methods: Eighty-five GPA patients were monitored for a median period of 3.1 years (range: 0.1–6.3). Circulating B cell subset frequencies were analyzed by flow cytometry determining the expression of CD19, CD38, and CD27. B cell subset frequencies at the time of inclusion of future-relapsing (F-R) and non-relapsing (N-R) patients were compared and related to relapse-free survival. Additionally, CD27+CD38hi B cells were assessed in urine and kidney biopsies from active anti-neutrophil cytoplasmic autoantibody-associated vasculitides (AAV) patients with renal involvement.Results: Within 1.6 years, 30% of patients experienced a relapse. The CD27+CD38hi B cell frequency at the time of inclusion was increased in F-R (median: 2.39%) compared to N-R patients (median: 1.03%; p = 0.0025) and a trend was found compared with the HCs (median: 1.33%; p = 0.08). This increased CD27+CD38hi B cell frequency at inclusion was correlated to decreased relapse-free survival in GPA patients. In addition, 74.7% of patients with an increased CD27+CD38hi B cell frequency (≥2.39%) relapsed during follow-up compared to 19.7% of patients with a CD27+CD38hi B cell frequency of

Loss of immune checkpoint (IC) Programmed Death-1 (PD-1) and PD-Ligand1 (PD-L1) expression has been implicated in the immunopathology of Giant Cell Arteritis (GCA). The contribution of the negative immune checkpoint V-domain Immunoglobulin-containing suppressor of T cell activation (VISTA) to GCA pathology has not yet been studied. The aim of our study was to investigate if expression of VISTA and other IC molecules by peripheral blood (PB) immune cells is modulated in GCA and at the site of vascular inflammation. In addition, we assessed the effect of VISTA-Ig engagement on in vitro CD4+ T helper (Th) lineage differentiation. To this end, frequencies of monocytes expressing CD80/86, PD-L1, PD-L2, and VISTA were determined in blood samples from 30 GCA patients and 18 matched healthy controls by flow cytometry. In parallel, frequencies of CD4+ cells expressing CD28, Cytotoxic T-Lymphocyte-associated antigen-4 (CTLA-4), PD-1, and VISTA were determined. Immunohistochemistry was employed to detect VISTA, PD-1, and PD-L1-expressing cells in temporal artery biopsies (TABs) diagnostic of GCA. Furthermore, the effect of VISTA-Ig on in vitro CD4+ Th lineage differentiation in patients and controls was determined. Our study shows that frequencies of CD80/CD86+ and VISTA+ monocytes were decreased in treated GCA patients only. Moreover, proportions of PD-1+ and VISTA+ Th cells were significantly decreased in GCA patients. Clear infiltration of VISTA+, PD1+, and PD-L1+ cells was seen in GCA TABs. Finally, VISTA-Ig engagement failed to suppress Th1, Th17, and Tfh lineage development in GCA. Our results indicate that decreased expression of VISTA may facilitate development of pathogenic Th1 and Th17 cells in GCA.

Autoreactive B cells are key drivers of pathogenic processes in autoimmune diseases by the production of autoantibodies, secretion of cytokines, and presentation of autoantigens to T cells. However, the mechanisms that underlie the development of autoreactive B cells are not well understood. Here, we review recent studies leveraging novel techniques to identify and characterize (auto)antigen-specific B cells. The insights gained from such studies pertaining to the mechanisms involved in the escape of tolerance checkpoints and the activation of autoreactive B cells are discussed. In addition, we briefly highlight potential therapeutic strategies to target and eliminate autoreactive B cells in autoimmune diseases.

Abstract High mobility group box 1 (HMGB1) is a nuclear DNA binding protein that acts as an alarmin when secreted. HMGB1 is increased in systemic lupus erythematosus and might represent a potential therapeutic target. We investigated whether treatment with an anti-HMGB1 antibody affects the development of lupus nephritis in MRL/lpr mice. Seven-week-old MRL/lpr mice were injected intraperitoneally twice weekly for 10 wks with 50 µg monoclonal anti-HMGB1 (2G7, mouse IgG2b) (n = 12) or control antibody (n = 11). Control MRL/MPJ mice (n = 10) were left untreated. Every 2 wks, blood was drawn and urine was collected at wk 7, 11 and 17. Mice were sacrificed at 17 wks for complete disease evaluation. Plasma HMGB1 and anti-HMGB1 levels were increased in MRL/lpr mice compared with control MRL/MPJ mice. There were no differences in albuminuria, urine HMGB1 and plasma levels of complement C3, anti-dsDNA and proinflammatory cytokines between untreated and treated mice at any time point. Lupus nephritis of mice treated with anti-HMGB1 monoclonal antibody (mAb) was classified as class III (n = 3) and class IV (n = 9), while mice treated with control mAb were classified as class II (n = 4), class III (n = 2) and class IV (n = 5). IgG and C3 deposits in kidneys were similar in mice treated with anti-HMGB1 mAb or control mAb. In conclusion, treatment with monoclonal anti-HMGB-1 antibody 2G7 does not affect development of lupus nephritis, disease progression or proinflammatory cytokine levels in MRL/lpr mice. This result indicates that blocking of HMGB1 by this neutralizing antibody does not affect lupus nephritis in MRL/lpr mice.

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28− T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a~24-2 cluster, in which all are predicted to bind to the 3′UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a~24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3′UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3′UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression.

Rodent models of both aging and obesity are characterized by inflammation in specific brain regions, notably the corpus callosum, fornix, and hypothalamus. Microglia, the resident macrophages of the central nervous system, are important for brain development, neural support, and homeostasis. However, the effects of diet and lifestyle on microglia during aging are only partly understood. Here, we report alterations in microglia phenotype and functions in different brain regions of mice on a high-fat diet (HFD) or low-fat diet (LFD) during aging and in response to voluntary running wheel exercise. We compared the expression levels of genes involved in immune response, phagocytosis, and metabolism in the hypothalamus of 6-month-old HFD and LFD mice. We also compared the immune response of microglia from HFD or LFD mice to peripheral inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS). Finally, we investigated the effect of diet, physical exercise, and caloric restriction (40% reduction compared to ad libitum intake) on microglia in 24-month-old HFD and LFD mice. Changes in diet caused morphological changes in microglia, but did not change the microglia response to LPS-induced systemic inflammation. Expression of phagocytic markers (i.e., Mac-2/Lgals3, Dectin-1/Clec7a, and CD16/CD32) in the white matter microglia of 24-month-old brain was markedly decreased in calorically restricted LFD mice. In conclusion, LFD resulted in reduced activation of microglia, which might be an underlying mechanism for the protective role of caloric restriction during aging-associated decline.

Age-associated changes in the immune system including alterations in surface protein expression are thought to contribute to an increased susceptibility for autoimmune diseases. The balance between the expression of coinhibitory and costimulatory surface protein molecules, also known as immune checkpoint molecules, is crucial in fine-tuning the immune response and preventing autoimmunity. The activation of specific inhibitory signaling pathways allows cancer cells to evade recognition and destruction by the host immune system. The use of immune checkpoint inhibitors (ICIs) to treat cancer has proven to be effective producing durable antitumor responses in multiple cancer types. However, one of the disadvantages derived from the use of these agents is the appearance of inflammatory manifestations termed immune-related adverse events (irAEs). These irAEs are often relatively mild, but more severe irAEs have been reported as well including several forms of vasculitis. In this article, we argue that age-related changes in expression and function of immune checkpoint molecules lead to an unstable immune system, which is prone to tolerance failure and autoimmune vasculitis development. The topic is introduced by a case report from our hospital describing a melanoma patient treated with ICIs and who subsequently developed biopsy-proven giant cell arteritis. Following this case report, we present an in-depth review on the role of immune checkpoint pathways in the development and progression of autoimmune vasculitis and its relation with an aging immune system.

B cells are central to the pathogenesis of granulomatosis with polyangiitis (GPA), exhibiting both (auto)antibody-dependent and -independent properties. Class-switched memory B cells in particular are a major source of pathogenic autoantibodies. These cells are characterized by high expression levels of Kv1.3 potassium channels, which may offer therapeutic potential for Kv1.3 blockade. In this study, we investigated the effect of the highly potent Kv1.3 blocker ShK-186 on B cell properties in GPA in vitro. Circulating B cell subsets were determined from 33 GPA patients and 17 healthy controls (HCs). Peripheral blood mononuclear cells (PBMCs) from GPA patients, and HCs were stimulated in vitro in the presence and absence of ShK-186. The production of total and antineutrophil cytoplasmic antibodies targeting proteinase 3 (PR3-ANCA) IgG was analyzed by enzyme-linked immunosorbent assay and Phadia EliA, respectively. In addition, effects of ShK-186 on B cell proliferation and cytokine production were determined by flow cytometry. The frequency of circulating switched and unswitched memory B cells was decreased in GPA patients as compared to HC. ShK-186 suppressed the production of both total and PR3-ANCA IgG in stimulated PBMCs. A strong decrease in production of tumor necrosis factor alpha (TNFα), interleukin (IL)-2, and interferon gamma was observed upon ShK-186 treatment, while effects on IL-10 production were less pronounced. As such, ShK-186 modulated the TNFα/IL-10 ratio among B cells, resulting in a relative increase in the regulatory B cell pool. ShK-186 modulates the effector functions of B cells in vitro by decreasing autoantibody and pro-inflammatory cytokine production. Kv1.3 channel blockade may hold promise as a novel therapeutic strategy in GPA and other B cell-mediated autoimmune disorders.

Patients with granulomatosis with polyangiitis (GPA) are prone to disease relapse. Currently, no good biomarkers are available to predict relapses in individual patients. This study aimed to determine whether patients at risk for relapse can be distinguished based on increased in vitro autoantibody production.Eighty-four proteinase 3 (PR3) anti-neutrophil cytoplasmic antibody (ANCA) positive GPA outpatients were prospectively monitored for up to two years and 32 healthy controls were included. At periodic intervals peripheral blood mononuclear cells were isolated, cultured and in vitro production of total and PR3-ANCA-specific IgG was determined. Moreover, serum ANCA titers were measured by indirect immunofluorescence.Sixteen patients (21%) relapsed during the follow-up period. At time of inclusion no significant differences were present for ANCA production between relapsing and non-relapsing patients. Samples before relapse exhibited increased serum ANCA titers and in vitro PR3-ANCA IgG levels compared with inclusion samples from non-relapsing patients. When evaluating changes over time, increasing serum ANCA titers were observed prior to relapse compared to a 1-year follow-up from non-relapsing patients. No significant change in in vitro PR3-ANCA levels occurred prior to relapse, compared to non-relapse patients.While differences were observed for the serum ANCA titer in relapsing and non-relapsing patients, monitoring in vitro PR3-ANCA IgG production does not improve relapse prediction in GPA patients.

Background. Naturally occurring substances from the flavanol and anthocyanin family of polyphenols have been proposed to exert beneficial effects in the course of obesity. We hypothesized that their effects on attenuating obesity-induced dyslipidemia as well as the associated inflammatory sequelae especially have health-promoting potential. Methods. Male C57BL/6J mice (n=52) received a control low-fat diet (LFD; 10 kcal% fat) for 6 weeks followed by 24 weeks of either LFD (n=13) or high-fat diet (HFD; 45 kcal% fat; n=13) or HFD supplemented with 0.1% w/w of the flavanol compound epicatechin (HFD+E; n=13) or an anthocyanin-rich bilberry extract (HFD+B; n=13). Energy substrate utilization was determined by indirect calorimetry in a subset of mice following the dietary switch and at the end of the experiment. Blood samples were collected at baseline and at 3 days and 4, 12, and 20 weeks after dietary switch and analyzed for systemic lipids and proinflammatory cytokines. Adipose tissue (AT) histopathology and inflammatory gene expression as well as hepatic lipid content were analyzed after sacrifice. Results. The switch from a LFD to a HFD lowered the respiratory exchange ratio and increased plasma cholesterol and hepatic lipid content. These changes were not attenuated by HFD+E or HFD+B. Furthermore, the polyphenol compounds could not prevent HFD-induced systemic rise of TNF-α levels. Interestingly, a significant reduction in Tnf gene expression in HFD+B mice was observed in the AT. Furthermore, HFD+B, but not HFD+E, significantly prevented the early upregulation of circulating neutrophil chemoattractant mKC. However, no differences in AT histopathology were observed between the HFD types. Conclusion. Supplementation of HFD with an anthocyanin-rich bilberry extract but not with the flavanol epicatechin may exert beneficial effects on the systemic early inflammatory response associated with diet-induced obesity. These systemic effects were transient and not observed after prolongation of HFD-feeding (24 weeks). On the tissue level, long-term treatment with bilberry attenuated TNF-α expression in adipose tissue.

Atherosclerosis is linked to inflammation. HDL protects against atherosclerotic cardiovascular disease, mainly by mediating cholesterol efflux and reverse cholesterol transport (RCT). The present study aimed to test the impact of acute inflammation as well as selected acute phase proteins on RCT with a macrophage-to-feces in vivo RCT assay using intraperitoneal administration of [3H]cholesterol-labeled macrophage foam cells. In patients with acute sepsis, cholesterol efflux toward plasma and HDL were significantly decreased (P < 0.001). In mice, acute inflammation (75 µg/mouse lipopolysaccharide) decreased [3H]cholesterol appearance in plasma (P < 0.05) and tracer excretion into feces both within bile acids (−84%) and neutral sterols (−79%, each P < 0.001). In the absence of systemic inflammation, overexpression of serum amyloid A (SAA, adenovirus) reduced overall RCT (P < 0.05), whereas secretory phospholipase A2 (sPLA2, transgenic mice) had no effect. Myeloperoxidase injection reduced tracer appearance in plasma (P < 0.05) as well as RCT (−36%, P < 0.05). Hepatic expression of bile acid synthesis genes (P < 0.01) and transporters mediating biliary sterol excretion (P < 0.01) was decreased by inflammation. In conclusion, our data demonstrate that acute inflammation impairs cholesterol efflux in patients and macrophage-to-feces RCT in vivo in mice. Myeloperoxidase and SAA contribute to a certain extent to reduced RCT during inflammation but not sPLA2. However, reduced bile acid formation and decreased biliary sterol excretion might represent major contributing factors to decreased RCT in inflammation.

ObjectiveDifferential gene expression in CD177+ and CD177- neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV).MethodsNeutrophils were isolated from healthy controls (HC) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177- subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177- subsets in microarray analysis were re-assessed using quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry.ResultsThe proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177- neutrophils resulted in 14 genes with fold change (fc) >3 difference in expression. Interestingly, 10 of these genes have been reported to change significantly in expression during neutrophil maturation, and most of these genes were granule protein (GP) coding genes. mRNA expression levels measured by RT-PCR of a number of these GP, and of PR3 and MPO were higher in the CD177- neutrophil subset in HC, however, particular granule protein amounts were comparable between CD177+ and CD177- neutrophil subsets. AAV patients had higher amounts of CD177+ neutrophils, but contrary to neutrophils from HC expression of GP-genes was increased, possibly due to activation.ConclusionThe neutrophil population can be distinguished by membrane expression of CD177 into subsets that are different in expression of GP mRNA but not in GP protein production. GP gene expression is also elevated in AAV patients, which is not explained by skewed distribution of CD177+ and CD177- subsets but may be associated with neutrophil activation during on-going inflammation.

A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, 12 and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10(-137)) in male mice; Ctnnbip1 (P = 6.42×10(-267)) and Tnfrsf8 (P = 5.42×10(-245)) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the Wnt-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the Wnt-signaling pathway that may be causally linked to tertiary lymphoid organ formation.

Renal aging is characterized by functional and structural changes like decreased glomerular filtration rate, and glomerular, tubular and interstitial damage. To gain insight in pathways involved in renal aging, we studied aged mouse strains and used genetic analysis to identify genes associated with aging phenotypes.Upon morphological screening in kidneys from 20-month-old mice from 26 inbred strains we noted intracapillary PAS-positive deposits. The severity of these deposits was quantified by scoring of a total of 50 glomeruli per section (grade 0-4). Electron microscopy and immunohistochemical staining for apoE, apoB, apoA-IV and perilipin-2 was performed to further characterize the lesions. To identify loci associated with these PAS-positive intracapillary glomerular deposits, we performed haplotype association mapping.Six out of 26 mouse strains showed glomerular PAS-positive deposits. The severity of these deposits varied: NOD(0.97), NZW(0.41), NON(0.30), B10(0.21), C3 H(0.9) and C57BR(0.7). The intracapillary deposits were strongly positive for apoE and weakly positive for apoB and apoA-IV. Haplotype association mapping showed a strong association with a 30-Kb haplotype block on Chr 1 within the Esrrg gene. We investigated 1 Mb on each site of this region, which includes the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3.By analyzing 26 aged mouse strains we found that some strains developed an intracapillary PAS and apoE-positive lesion and identified a small haplotype block on Chr 1 within the Esrrg gene to be associated with these lipoprotein deposits. The region spanning this haplotype block contains the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3, which are all highly expressed in the kidney. Esrrg might be involved in the evolvement of these glomerular deposits by influencing lipid metabolism and possibly immune reponses.

p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus examined expression and activation of p38MAPK isoforms during anti-glomerular basement membrane (GBM) nephritis. Therefore, p38α conditional knockout mice (MxCre-p38α(Δ/Δ)) were used to examine the role of p38α in anti-GBM induced nephritis. Both wild type and MxCre-p38α(Δ/Δ) mice developed acute renal failure over time. Histological examinations revealed a reduced monocyte influx and less tubular damage in MxCre-p38α(Δ/Δ) mice, whereas glomerular crescent formation and renal fibrosis was similar. Likewise, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar, but IL-8 was even up-regulated in MxCre-p38α(Δ/Δ) mice. In contrast, we could detect strong down-regulation of chemotactic cytokines such as CCL-2, -5 and -7, in the kidneys of MxCre-p38α(Δ/Δ) mice. In conclusion, p38α is the primary p38MAPK isoform expressed in anti-GBM nephritis and selectively affects inflammatory cell influx and tubular damage. Full protection from nephritis is however not achieved as renal failure and structural damage still occurs.

BackgroundAn Alternating high- cholesterol dietary regimen has proven to be beneficial when compared to daily high- cholesterol feeding. In the current study we explored whether the same strategy is applicable to a high- fat dietary regimen.ObjectiveTo investigate whether an alternating high- fat dietary regimen can effectively diminish insulin resistance, hepatic and renal inflammation and renal dysfunction as compared to a continuous high- fat diet.DesignFour groups of male ApoE*3Leiden mice (n=15) were exposed to different diet regimens for 20 weeks as follows: Group 1: low- fat diet (10 kcal% fat); Group 2: intermediate- fat diet (25 kcal% fat); Group 3: high- fat diet (45 kcal% fat) and Group 4: alternating- fat diet (10 kcal% fat for 4 days and 45 kcal% fat for 3 days in a week).ResultsCompared to high fat diet feeding, the alternating and intermediate- fat diet groups had reduced body weight gain and did not develop insulin resistance or albuminuria. In addition, in the alternating and intermediate- fat diet groups, parameters of tissue inflammation were markedly reduced compared to high fat diet fed mice.ConclusionBoth alternating and intermediate- fat feeding were beneficial in terms of reducing body weight gain, insulin resistance, hepatic and renal inflammation and renal dysfunction. Thus beneficial effects of alternating feeding regimens on cardiometabolic risk factors are not only applicable for cholesterol containing diets but can be extended to diets high in fat content.

Formation of advanced glycation endproducts (AGEs), endothelial dysfunction, and low-grade inflammation are intermediate pathways of hyperglycemia-induced vascular complications. We investigated the effect of benfotiamine on markers of these pathways in patients with type 2 diabetes and nephropathy.Patients with type 2 diabetes and urinary albumin excretion in the high-normal and microalbuminuric range (15-300 mg/24h) were randomized to receive benfotiamine (n = 39) or placebo (n = 43). Plasma and urinary AGEs (N(ε)-(carboxymethyl) lysine [CML], N(ε)-(Carboxyethyl) lysine [CEL], and 5-hydro-5-methylimidazolone [MG-H1]) and plasma markers of endothelial dysfunction (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble intercellular adhesion molecule-1 [sICAM-1], soluble E-selectin) and low-grade inflammation (high-sensitivity C-reactive protein [hs-CRP], serum amyloid-A [SAA], myeloperoxidase [MPO]) were measured at baseline and after 6 and 12 weeks.Compared to placebo, benfotiamine did not result in significant reductions in plasma or urinary AGEs or plasma markers of endothelial dysfunction and low-grade inflammation.Benfotiamine for 12 weeks did not significantly affect intermediate pathways of hyperglycemia-induced vascular complications. TRIAL REGRISTRATION: ClinicalTrials.gov NCT00565318.

INTRODUCTION: Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo. METHODS: Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed. RESULTS: In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4(+) monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC. CONCLUSIONS: In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation.

The etiology of anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) is unknown, but the association between infections and autoimmunity has been studied extensively. In 2004, a novel theory was proposed that could link infection and autoimmunity. This 'theory of autoantigen complementarity' was based on the serendipitous finding of antibodies against complementary-PR3 (cPR3) in patients with PR3-ANCA-associated vasculitis. cPR3 demonstrated homology to several bacterial proteins, and it was hypothesized that PR3-ANCA develop in response to anti-cPR3 antibodies, as a consequence of the anti-idiotypic network. These data have not been confirmed in other patient cohorts. We investigated the presence of anti-cPR3 antibodies in a Dutch cohort of PR3-ANCA-associated vasculitis patients. Anti-cPR3 reactivity was determined in serum using ELISA. Two separate batches of cPR3 were used to determine reactivity in two separate cohorts of PR3-ANCA-associated vasculitis patients. We found that anti-cPR3-reactivity was not increased in our PR3-ANCA-associated vasculitis patients, in comparison to control groups. Further research will be necessary to prove the concept of autoantigen complementarity in autoimmune diseases.

BACKGROUND: Alternate day calorie restriction (CR) has been shown to be almost as beneficial as daily CR. The question arises whether this concept is also applicable to alternating dietary composition. OBJECTIVE: To seek evidence that alternating high cholesterol (HC)-cholesterol-free (CON) Western diet can effectively diminish hepatic and renal inflammation and cardiovascular risk factors as compared with daily HC-supplemented Western diet. DESIGN: Four groups of ApoE*3Leiden mice, a humanized model for atherosclerosis, were subjected to different feeding treatments for 16 weeks. Mice were fed CON diet; CON diet with 1% w/w cholesterol (HC); alternate (ALT) diet regimen of CON (4 days) and HC (3 days); or CON diet supplemented with 0.43% (w/w) cholesterol (MC), with overall dietary cholesterol intake equal to ALT. Plasma was analyzed for cardiovascular risk factors, aorta for atherosclerotic lesion formation, and liver and kidney for inflammation. RESULTS: ALT diet but not MC was almost as effective as daily CON feeding in preventing disease development. Compared to HC, the ALT group showed 62% lower hepatic nuclear factor kappa B (NF-κB) activity (P