B lymphocyte responses in Parkinson's disease and their possible significance in disease progression.

Inflammation contributes to Parkinson's disease pathogenesis. We hypothesized that B lymphocytes are involved in Parkinson's disease progression. We measured antibodies to alpha-synuclein and tau in serum from patients with rapid eye movement sleep behaviour disorder (n = 79), early Parkinson's disease (n = 50) and matched controls (n = 50). Rapid eye movement sleep behaviour disorder cases were stratified by risk of progression to Parkinson's disease (low risk = 30, high risk = 49). We also measured B-cell activating factor of the tumour necrosis factor receptor family, C-reactive protein and total immunoglobulin G. We found elevated levels of antibodies to alpha-synuclein fibrils in rapid eye movement sleep behaviour disorder patients at high risk of Parkinson's disease conversion (ANOVA, P < 0.001) and lower S129D peptide-specific antibodies in those at low risk (ANOVA, P < 0.001). An early humoral response to alpha-synuclein is therefore detectable prior to the development of Parkinson's disease. Peripheral B lymphocyte phenotyping using flow cytometry in early Parkinson's disease patients and matched controls (n = 41 per group) revealed reduced B cells in Parkinson's disease, particularly in those at higher risk of developing an early dementia [t(3) = 2.87, P = 0.01]. Patients with a greater proportion of regulatory B cells had better motor scores [F(4,24) = 3.612, P = 0.019], suggesting they have a protective role in Parkinson's disease. In contrast, B cells isolated from Parkinson's disease patients at higher risk of dementia had greater cytokine (interleukin 6 and interleukin 10) responses following in vitro stimulation. We assessed peripheral blood lymphocytes in alpha-synuclein transgenic mouse models of Parkinson's disease: they also had reduced B cells, suggesting this is related to alpha-synuclein pathology. In a toxin-based mouse model of Parkinson's disease, B-cell deficiency or depletion resulted in worse pathological and behavioural outcomes, supporting the conclusion that B cells play an early protective role in dopaminergic cell loss. In conclusion, we found changes in the B-cell compartment associated with risk of disease progression in rapid eye movement sleep behaviour disorder (higher alpha-synuclein antibodies) and early Parkinson's disease (lower levels of B lymphocytes that were more reactive to stimulation). Regulatory B cells play a protective role in a mouse model, potentially by attenuating inflammation and dopaminergic cell loss. B cells are therefore likely to be involved in the pathogenesis of Parkinson's disease, albeit in a complex way, and thus warrant consideration as a therapeutic target.

B Lymphocyte-Derived CCL7 Augments Neutrophil and Monocyte Recruitment, Exacerbating Acute Kidney Injury.

Acute kidney injury (AKI) is a serious condition affecting one fifth of hospital inpatients. B lymphocytes have immunological functions beyond Ab production and may produce cytokines and chemokines that modulate inflammation. In this study, we investigated leukocyte responses in a mouse model of AKI and observed an increase in circulating and kidney B cells, particularly a B220low subset, following AKI. We found that B cells produce the chemokine CCL7, with the potential to facilitate neutrophil and monocyte recruitment to the injured kidney. Siglec-G-deficient mice, which have increased numbers of B220low innate B cells and a lower B cell activation threshold, had increased Ccl7 transcripts, increased neutrophil and monocyte numbers in the kidney, and more severe AKI. CCL7 blockade in mice reduced myeloid cell infiltration into the kidney and ameliorated AKI. In two independent cohorts of human patients with AKI, we observed significantly higher CCL7 transcripts compared with controls, and in a third cohort, we observed an increase in urinary CCL7 levels in AKI, supporting the clinical importance of this pathway. Together, our data suggest that B cells contribute to early sterile inflammation in AKI via the production of leukocyte-recruiting chemokines.

GM-CSF Calibrates Macrophage Defense and Wound Healing Programs during Intestinal Infection and Inflammation.

Macrophages play a central role in intestinal immunity, but inappropriate macrophage activation is associated with inflammatory bowel disease (IBD). Here, we identify granulocyte-macrophage colony stimulating factor (GM-CSF) as a critical regulator of intestinal macrophage activation in patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis. We find that GM-CSF drives the maturation and polarization of inflammatory intestinal macrophages, promoting anti-microbial functions while suppressing wound-healing transcriptional programs. Group 3 innate lymphoid cells (ILC3s) are a major source of GM-CSF in intestinal inflammation, with a strong positive correlation observed between ILC or CSF2 transcripts and M1 macrophage signatures in IBD mucosal biopsies. Furthermore, GM-CSF-dependent macrophage polarization results in a positive feedback loop that augmented ILC3 activation and type 17 immunity. Together, our data reveal an important role for GM-CSF-mediated ILC-macrophage crosstalk in calibrating intestinal macrophage phenotype to enhance anti-bacterial responses, while inhibiting pro-repair functions associated with fibrosis and stricturing, with important clinical implications.

Macrophage metabolic reprogramming presents a therapeutic target in lupus nephritis.

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1ß, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1ß and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis.

Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis.

Inflammatory bowel disease is a chronic, relapsing condition with two subtypes, Crohn's disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWASs) in UC implicate a FCGR2A variant that alters the binding affinity of the antibody receptor it encodes, FcγRIIA, for immunoglobulin G (IgG). Here, we aimed to understand the mechanisms whereby changes in FcγRIIA affinity would affect inflammation in an IgA-dominated organ. We found a profound induction of anti-commensal IgG and a concomitant increase in activating FcγR signaling in the colonic mucosa of UC patients. Commensal-IgG immune complexes engaged gut-resident FcγR-expressing macrophages, inducing NLRP3- and reactive-oxygen-species-dependent production of interleukin-1ß (IL-1ß) and neutrophil-recruiting chemokines. These responses were modulated by the FCGR2A genotype. In vivo manipulation of macrophage FcγR signal strength in a mouse model of UC determined the magnitude of intestinal inflammation and IL-1ß-dependent type 17 immunity. The identification of an important contribution of IgG-FcγR-dependent inflammation to UC has therapeutic implications.

Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense.

FcγRIIb inhibits immune complex-induced VEGF-A production and intranodal lymphangiogenesis.

IgG immune complexes (ICs) are generated during immune responses to infection and self-antigen and have been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Their role, and that of the fragment crystallizable (Fc) receptors that bind them, in driving local inflammation is not fully understood. Low affinity-activating Fcγ receptors (FcγRs) that bind immune complexes are controlled by a single inhibitory receptor, FcγRIIb (CD32b). We investigated whether FcγR cross-linking by IC might induce VEGF-A and lymph node lymphangiogenesis. Murine macrophages and dendritic cells (DCs) stimulated with ICs produced VEGF-A, and this was inhibited by coligation of FcγRIIb. Similarly, IC-induced VEGF-A production by B cells was inhibited by FcγRIIb. In vivo, IC generation resulted in VEGF-A-dependent intranodal lymphangiogenesis and increased DC number. We sought to determine the relevance of these findings to autoimmunity because elevated serum VEGF-A has been observed in patients with SLE; we found that lymphangiogenesis and VEGF-A were increased in the lymph nodes of mice with collagen-induced arthritis and SLE. In humans, a SLE-associated polymorphism (rs1050501) results in a dysfunctional FcγRIIB(T232) receptor. Monocyte-derived macrophages from subjects with the FcγRIIB(T/T232) genotype showed increased FcγR-mediated VEGF-A production, demonstrating a similar process is likely to occur in humans. Thus, ICs contribute to inflammation through VEGF-A-driven lymph node lymphangiogenesis, which is controlled by FcγRIIb. These findings have implications for the pathogenesis, and perhaps future treatment, of autoimmune diseases.

Immune complexes stimulate CCR7-dependent dendritic cell migration to lymph nodes.

Antibodies are critical for defense against a variety of microbes, but they may also be pathogenic in some autoimmune diseases. Many effector functions of antibodies are mediated by Fcγ receptors (FcγRs), which are found on most immune cells, including dendritic cells (DCs)-important antigen-presenting cells that play a central role in inducing antigen-specific tolerance or immunity. Following antigen acquisition in peripheral tissues, DCs migrate to draining lymph nodes via the lymphatics to present antigen to T cells. Here we demonstrate that FcγR engagement by IgG immune complexes (ICs) stimulates DC migration from peripheral tissues to the paracortex of draining lymph nodes. In vitro, IC-stimulated mouse and human DCs showed greater directional migration in a chemokine (C-C) ligand 19 (CCL19) gradient and increased chemokine (C-C) receptor 7 (CCR7) expression. Using intravital two-photon microscopy, we observed that local administration of IC resulted in dermal DC mobilization. We confirmed that dermal DC migration to lymph nodes depended on CCR7 and increased in the absence of the inhibitory receptor FcγRIIB. These observations have relevance to autoimmunity because autoantibody-containing serum from humans with systemic lupus erythematosus (SLE) and from a mouse model of SLE also increased dermal DC migration in vivo, suggesting that this process may occur in lupus, potentially driving the inappropriate localization of autoantigen-bearing DCs.

Evidence of NLRP3-inflammasome activation in rheumatoid arthritis (RA); genetic variants within the NLRP3-inflammasome complex in relation to susceptibility to RA and response to anti-TNF treatment.

BACKGROUND: The NLRP3-inflammasome, implicated in the pathogenesis of several inflammatory disorders, has been analysed in rheumatoid arthritis (RA). METHODS: Relative gene expression of NLRP3-inflammasome components was characterised in PBMCs of 29 patients receiving infliximab. A total of 1278 Caucasian patients with RA from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort receiving tumour necrosis factor (TNF) antagonists (infliximab, adalimumab and etanercept) were genotyped for 34 single nucleotide polymorphisms (SNPs), spanning the genes NLRP3, MEFV and CARD8. Regression analyses were performed to test for association between genotype and susceptibility and treatment response (disease activity score across 28 joints (DAS28) and EULAR improvement criteria) at 6 months, with secondary expression quantitative trait loci (eQTL) analyses. RESULTS: At baseline, gene expression of ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1 were significantly higher compared with controls whereas CARD8 was lower in the patients. Caspase-1 and interleukin-18 levels were significantly raised in patients with RA. SNPs in NLRP3 showed association with RA susceptibility and EULAR response to anti-TNF in the BRAGGSS cohort, and in monocytes but not B cells, in eQTL analysis of 283 healthy controls. CARD8 SNPs were associated with RA susceptibility and DAS28 improvement in response to anti-TNF and eQTL effects in monocytes and B cells. CONCLUSIONS: This study found evidence of modulation of the NLRP3-inflammasome in patients with RA prior to receiving infliximab and some evidence of association for SNPs at NLRP3 and CARD8 loci with RA susceptibility and response to anti-TNF. The SNPs associated with susceptibility/response are not the main eQTL variants for either locus, and the associations with treatment response require replication in an independent cohort.

Magnetic resonance imaging with hyperpolarized [1,4-(13)C2]fumarate allows detection of early renal acute tubular necrosis.

Acute kidney injury (AKI) is a common and important medical problem, affecting 10% of hospitalized patients, and it is associated with significant morbidity and mortality. The most frequent cause of AKI is acute tubular necrosis (ATN). Current imaging techniques and biomarkers do not allow ATN to be reliably differentiated from important differential diagnoses, such as acute glomerulonephritis (GN). We investigated whether (13)C magnetic resonance spectroscopic imaging (MRSI) might allow the noninvasive diagnosis of ATN. (13)C MRSI of hyperpolarized [1,4-(13)C(2)]fumarate and pyruvate was used in murine models of ATN and acute GN (NZM2410 mice with lupus nephritis). A significant increase in [1,4-(13)C(2)]malate signal was identified in the kidneys of mice with ATN early in the disease course before the onset of severe histological changes. No such increase in renal [1,4-(13)C(2)]malate was observed in mice with acute GN. The kidney [1-(13)C]pyruvate/[1-(13)C]lactate ratio showed substantial variability and was not significantly decreased in animals with ATN or increased in animals with GN. In conclusion, MRSI of hyperpolarized [1,4-(13)C(2)]fumarate allows the detection of early tubular necrosis and its distinction from glomerular inflammation in murine models. This technique may have the potential to identify a window of therapeutic opportunity in which emerging therapies might be applied to patients with ATN, reducing the need for acute dialysis with its attendant morbidity and cost.

Vitamin D3 down-regulates intracellular Toll-like receptor 9 expression and Toll-like receptor 9-induced IL-6 production in human monocytes.

OBJECTIVE: To determine whether vitamin D(3) modulates monocytic expression of intracellular Toll-like receptors (TLRs) 3, 7 and 9. METHODS: Human monocytes were isolated from peripheral blood and cultured with 100 nM vitamin D(3) for 24, 48 and 72 h. Expression of CD14 and TLR2, TLR3, TLR4, TLR7 and TLR9 were examined by flow cytometry. Monocytes exposed to vitamin D(3) for 48 h were then stimulated with a TLR9 agonist for a further 24 h. The level of IL-6 secretion was measured by ELISA. RESULTS: CD14 was up-regulated, whereas TLR2, TLR4 and TLR9 expression was down-regulated by vitamin D(3) exposure in a time-dependent manner. TLR3 expression was unaffected by vitamin D(3) and there was no measurable expression of TLR7 on the monocytes. TLR9-induced IL-6 production was impaired in monocytes treated with vitamin D(3) compared with untreated cells. CONCLUSION: The intracellular TLRs are differentially regulated by vitamin D(3), with TLR9 being down-regulated by vitamin D(3) exposure whereas TLR3 was unaffected. This decreased TLR9 expression in monocytes had a downstream functional effect as these cells subsequently secreted less IL-6 in response to TLR9 challenge. This may have significant biological relevance and may be a factor in the association of vitamin D deficiency with susceptibility to autoimmune disease.

NOD-like receptors and inflammation.

The nucleotide-binding and oligomerization domain, leucine-rich repeat (also known as NOD-like receptors, both abbreviated to NLR) family of intracellular pathogen recognition receptors are increasingly being recognized to play a pivotal role in the pathogenesis of a number of rare monogenic diseases, as well as some more common polygenic conditions. Bacterial wall constituents and other cellular stressor molecules are recognized by a range of NLRs, which leads to activation of the innate immune response and upregulation of key proinflammatory pathways, such as IL-1beta production and translocation of nuclear factor-kappaB to the nucleus. These signalling pathways are increasingly being targeted as potential sites for new therapies. This review discusses the role played by NLRs in a variety of inflammatory diseases and describes the remarkable success to date of these therapeutic agents in treating some of the disorders associated with aberrant NLR function.

Fifth International Congress on Familial Mediterranean Fever and Systemic Autoinflammatory Diseases.

The Fifth International Congress on Familial Mediterranean Fever and Systemic Autoinflammatory Diseases (Rome, Italy, 4-8 April, 2008) reviewed developments in the field of innate immunity and discussed their relevance to the pathogenesis of associated diseases. The meeting gathered over 300 participants from 32 countries. New modes of inheritance of autoinflammatory diseases, animal models, novel related genes and the remarkable efficacy of IL-1beta blockage in most of these diseases were emphasized.