IMMUNE MECHANISMS ASSOCIATED TO NEUROINFLAMMATION IN ALZHEIMER¿S DISEASE

La patogenesi dell' Alzheimer (AD) non \ue8 nota, tuttavia \ue8 sempre pi\uf9 chiaro che l\u2019 infiammazione, processo associato all\u2019insorgenza di numerose malattie neurodegenerative del sistema nervoso centrale, svolga un ruolo in tale patologia. L\u2019infiammazione \ue8 una componente chiave della risposta immunitaria innata. L\u2019immunit\ue0 innata \ue8 un sistema altamente conservato che protegge l\u2019ospite dalle infezioni in maniera aspecifica. Sebbene questo sistema rappresenti una risposta efficace e potente agli stimoli acuti \ue8 necessario che sia finemente regolato: una deregolazione o un\u2019attivazione cronica possono infatti avere effetti dannosi per l\u2019ospite. L\u2019infiammazione cronica \ue8 stata implicata non solo in malattie periferiche ma anche in malattie neurodegenerative del sistema nervoso centrale come l\u2019Alzheimer. L\u2019ipotesi di questo lavoro \ue8 stata che l\u2019infiammazione rappresenti un fattore negativo per la malattia di Alzheimer e che i meccanismi che concorrono a regolare la risposta infiammatoria siano quantitativamente e funzionalmente compromessi negli AD rispetto agli MCI e HC. Uno dei meccanismi di regolazione della tolleranza immunologica \ue8 rappresentato dai linfociti Treg. I risultati presentati indicano che lo sviluppo della patologia di AD \ue8 associato ad una diminuzione del numero di Treg circolanti e in particolare della percentuale di Treg naive. Quest\u2019alterazione quantitativa \ue8 associata ad un\u2019alterazione qualitativa quale un\u2019aumentata proliferazione amiloide- specifica e ad una ridotta capacit\ue0 dei Treg di sopprimere tale proliferazione. L\u2019analisi del pathway PD1-PDL1, in grado di controllare la risposta infiammatoria mediante produzione di IL-10 e induzione di apoptosi antigene-specifica, ha mostrato una diminuzione dell\u2019espressione di PD-1 sui linfociti T CD4+ dei pazienti AD e MCI rispetto ai controlli sani. I risultati mostrano inoltre una diminuzione significativa della produzione di IL-10 da parte di CD14+PD-L1+. La down-regolazione di questi meccanismi osservata nei pazienti AD e MCI risulta in un aumento della proliferazione dei linfociti T stimolati alla \u3b2A. Il ruolo chiave svolto dall\u2019interazione PD-1/PD-L1 nell\u2019indurre l\u2019apoptosi dei linfociti T CD4+ specifici per \u3b2A \ue8 confermato dall\u2019osservazione che l\u2019apoptosi \ue8 bloccata preincubando queste cellule con un anticorpo bloccante anti- PD-L1. Lo studio delle sottopopolazioni linfocitarie nelle forme di AD rispetto alla forma MCI e agli HC ha inoltre evidenziato che nei pazienti AD non solo vi \ue8 un\u2019alterazione nei meccanismi di tolleranza immunologica ma anche uno shift nel differenziamento del linfociti T verso un fenotipo infiammatorio di tipo Th-17 e Th-9. I risultati hanno, infatti, mostrato un aumento della produzione delle citochine infiammatorie (IL-21, IL-23, IL-6) e dei fattori di trascrizione (RORc/gt) coinvolti nel differenziamento dei Th-17 cos\uec come delle citochine effettrici (IL-21 e IL-22) prodotte da tali cellule nei pazienti AD rispetto agli MCI e agli HC. In particolare, IL-9, la citochina effettrice prodotta dalle Th-9, \ue8 significativamente aumentata nei pazienti AD, indicando che oltre ai Th-17 anche i Th-9 specifici per la \u3b2A sono upregolati negli AD e Th-9 (IL-21 e IL-22). In conclusione la compromissione della risposta immunitaria, con una profonda inclinazione a favore di risposte effettrici e infiammatorie, sembra svolgere un ruolo chiave in questa patologia.The etiology of Alzheimer\u2019 s disease (AD) is still unresolved, even if it is becoming clearer that inflammation, a process associated to the onset of several neurodegenerative disorders, plays a central role in this disease. Inflammation is a key component of innate immune system. Innate immunity is a very highly conserved system that protects the host from infections in a non-specific manner. Even if this system provides a powerful response to a range of insults it must be tightly regulated: deregulation and chronic activation can have detrimental effects on the host. Chronic inflammation has been involved not only in peripheral diseases but also in neurodegenerative diseases of the central nervous system, like Alzheimer\u2019s disease. Our working hypothesis is that inflammation plays a negative role in this pathology and that the mechanisms regulating the inflammatory responses are functional compromised in AD patients compared to Mild cognitive impairment (MCI) and to healthy controls (HC). One of the main way in which immunologic tolerance is modulated is through T regulatory cells (Treg). Our results indicate that the development of AD is associated with a reduction of circulating T reg na\uefve cells, the subpopulation of Treg cells endowed with the strongest suppressive ability. These quantitative changes are associated with qualitative changes, summarized as an increase of A\u3b2-specific proliferation and a reduced ability of Treg to suppress such proliferation. The analysis of the PD-1/PD-L1 pathway, which modulates the balance between inflammation and tolerance by inducing IL-10 production and apoptosis of antigen-specific cells, shows a decrease of PD-1 expressing CD4+ T cells in AD and MCI compared to HC as well as a decrease of PD-L1- expressing and IL-10-producing CD14+ cells. The impairment of the PD-1/PD-L1 pathway in AD patients results in reduced IL-10 production and diminished apoptosis of A\u3b2-specific CD4+ T lymphocytes. The central role performed by PD-1/PD-L1 pathway in inducing the apoptosis of Abeta-specific T cells is confirmed by the observation that apoptosis is inhibited pre-incubating lymphocytes with a PD-L1-specific blocking antibody. The analysis of lymphocytes subpopulations in AD and MCI compared to controls highlight that in AD patients not only an alteration of immunological tolerance is present but also a shift in the differentiation of T lymphocytes towards an inflammatory phenotype Th-9 and Th-17. Our results showed indeed that cytokines (IL-21, IL-23, IL-6) and transcription factor (RORc/gt) involved in the differentiation of Th-17, as well as cytokines (IL-21, IL-22) produced by these cells are all augmented in AD compared to MCI and HC. Notably, IL-9, the effector cytokine produced by Th-9 cells, was significantly increased as well in AD patients, indicating that, beside Th-17, A\u3b2- specific Th-9 lymphocytes are upregulated in AD. In conclusion the impairment of the immune response, with a profound skewing favoring inflammatory and effector responses, seem to play a pivotal role in this pathology

IL-33 and its decoy sST2 in patients with Alzheimer's disease and mild cognitive impairment

BACKGROUND: Interleukin-33 is a cytokine endowed with pro- and anti-inflammatory properties that plays a still poorly defined role in the pathogenesis of a number of central nervous system (CNS) conditions including Alzheimer's disease (AD). We analyzed this cytokine and its decoy receptor sST2 in Alzheimer's disease (AD) and mild cognitive impairment (MCI). METHOD: IL-33 and sST2 were analyzed in serum and CSF of AD and MCI patients, comparing the results to those obtained in age-matched healthy controls (HC). Because of the ambiguous role of IL-33 in inflammation, the concentration of both inflammatory (IL-1\u3b2 and IL-6) and anti-inflammatory (IL-10) cytokines was analyzed as well in serum and cerebrospinal fluid (CSF) of the same individuals. Finally, the effect of IL-33 on in vitro A\u3b242-stimulated monocytes of AD, MCI, and HC individuals was examined. RESULTS: As compared to HC, (1) IL-33 was significantly decreased in serum and CSF of AD and MCI, (2) sST2 was increased in serum of AD and MCI but was undetectable in CSF, (3) serum and CSF IL-1\u3b2 concentration was significantly increased and that of IL-10 was reduced in AD and MCI, whereas no differences were observed in IL-6. In vitro addition of IL-33 to LPS+A\u3b2 42-stimulated monocytes downregulated IL-1\u3b2 generation in MCI and HC, but not in AD, and stimulated IL-10 production in HC alone. IL-33 addition also resulted in a significant reduction of NF-kB nuclear translocation in LPS+A\u3b242-stimulated monocytes of HC alone. CONCLUSIONS: These data support the hypothesis that IL-33 plays a complex anti-inflammatory role that is lost in AD- and MCI-associated neuroinflammation; results herein also suggest a possible use of IL-33 as a novel therapeutic approach in AD and MCI

Endoplasmic Reticulum Associated Aminopeptidase 2 (ERAP2) is released in the secretome of activated MDMs and reduces in vitro HIV-1 infection

Background: Haplotype-specific alternative splicing of the endoplasmic reticulum (ER) aminopeptidase type 2 (ERAP2) gene results in either full-length (FL, haplotype A) or alternatively spliced (AS, haplotype B) mRNA. HapA/HapA homozygous (HomoA) subjects show a reduced susceptibility to HIV-1 infection, probably secondary to the modulation of the antigen processing/presenting machinery. ERAP1 was recently shown to be secreted from the plasma membrane in response to activation; we investigated whether ERAP2 can be released as well and if the secreted form of this enzyme retains its antiviral function. Methods: Human monocyte derived macrophages (MDMs) were differentiated from peripheral blood mononuclear cells (PBMCs) isolated from 6 HomoA healthy controls and stimulated with IFN\u3b3 and LPS. ERAP2-FL secretion was evaluated by mass spectrometry. PBMCs (14 HomoA and 16 HomoB) and CD8-depleted PBMCs (CD8-PBMCs) (4 HomoA and 4 HomoB) were in vitro HIV-infected in the absence/presence of recombinant human ERAP2-FL (rhERAP2) protein; p24 viral antigen quantification was used to assess viral replication. IFN\u3b3 and CD69 mRNA expression, as well as the percentage of perforin-producing CD8+ T Lymphocytes, were analyzed 3 and 7-days post in vitro HIV-1-infection, respectively. The effect of rhERAP2 addition in cell cultures on T cell apoptosis, proliferation, activation, and maturation was evaluated as well on 24 h-stimulated PBMCs. Results: ERAP2 can be secreted from human MDMs in response to IFN\u3b3/LPS stimulation. Notably, the addition of rhERAP2 to PBMC and CD8-PBMC cultures resulted in the reduction of viral replication, though these differences were statistically significant only in PBMCs (p < 0.05 in both HomoA and HomoB). This protective effect was associated with an increase in IFN\u3b3 and CD69 mRNA expression and in the percentage of perforin-expressing CD107+CD8+ cells. RhERAP2 addition also resulted in an increase in CD8+ activated lymphocyte (CD25+HLA-DRII+) and Effector Memory/Terminally differentiated CD8+ T cells ratio. Conclusions: This is the first report providing evidence for the release of ERAP2 in the secretome of immunocompetent cells. Data herein also indicate that exogenous ERAP2-FL exerts its protective function against HIV-1 infection, even in HomoB subjects who do not genetically produce it. Presumably, this defensive extracellular feature is only partially dependent on immune system modulation

Influence of a high‐impact multidimensional rehabilitation program on the gut microbiota of patients with multiple sclerosis

Multiple sclerosis (MS) is a neurodegenerative inflammatory condition mediated by autoreactive immune processes. Due to its potential to influence host immunity and gut‐brain communication, the gut microbiota has been suggested to be involved in the onset and progression of MS. To date, there is no definitive cure for MS, and rehabilitation programs are of the utmost importance, especially in the later stages. However, only a few people generally participate due to poor support, knowledge, and motivation, and no information is available on gut microbiota changes. Herein we evaluated the potential of a brief high‐impact multidimensional rehabilitation program (B‐HIPE) in a leisure environment to affect the gut microbiota, mitigate MS symptoms and improve quality of life. B‐HIPE resulted in modulation of the MS‐typical dysbiosis, with reduced levels of pathobionts and the replenishment of beneficial short‐chain fatty acid producers. This partial recovery of a eubiotic profile could help counteract the inflammatory tone typically observed in MS, as supported by reduced circulating lipopolysaccharide levels and decreased populations of pro‐inflammatory lymphocytes. Improved physical performance and fatigue relief were also found. Our findings pave the way for integrating clinical practice with holistic approaches to mitigate MS symptoms and improve patients’ quality of life

Immune and Imaging Correlates of Mild Cognitive Impairment Conversion to Alzheimer's Disease

Amnestic mild cognitive impairment (aMCI) conversion to Alzheimer's disease (AD) is seen in a sizable portion of aMCI patients; correlates predicting such conversion are poorly defined but neuroinflammation and the reactivation of chronic viral infections are suspected to play a role in this phenomenon. We analyzed these aspects in two homogeneous groups of aMCI who did or did not convert to AD over a 24-months period. Results showed that at baseline in those aMCI individuals who did not convert to AD: 1) A\uce\ub21-42stimulated production of the pro-inflammatory cytokines IL6 and IL1\uce\ub2 by CD14+cells was significantly reduced (p = 0.01), 2) CD14+/IL-33+cells were increased (p = 0.0004); 3) MFI of TLR8 and TLR9 was significantly increased, and 4) better preserved hippocampus volumes were observed and correlated with IL33+/CD14+cells. Notably, A\uce\ub21-42stimulated production of the antiviral cytokine IFN-\uce\ubb was increased as well in non-AD converters, although with a borderline statistical significance (p = 0.05). Data herein indicating that proinflammatory cytokines are reduced, whereas IFN-\uce\ubb production and TLR8 and 9 MFI are augmented in those aMCI in whom AD conversion is not observed suggest that the ability to mount stronger antiviral response within an antiiflammatory milieu associates with lack of AD conversion

The NLRP3 and NLRP1 Inflammasomes are Activated in Alzheimer’s Disease

Background Interleukin-1 beta (IL-1\u3b2) and its key regulator, the inflammasome, are suspected to play a role in the neuroinflammation observed in Alzheimer\u2019s disease (AD); no conclusive data are nevertheless available in AD patients. Results mRNA for inflammasome components (NLRP1, NLRP3, PYCARD, caspase 1, 5 and 8) and downstream effectors (IL-1\u3b2, IL-18) was up-regulated in severe and MILD AD. Monocytes co-expressing NLRP3 with caspase 1 or caspase 8 were significantly increased in severe AD alone, whereas those co-expressing NLRP1 and NLRP3 with PYCARD were augmented in both severe and MILD AD. Activation of the NLRP1 and NLRP3 inflammasomes in AD was confirmed by confocal microscopy proteins co-localization and by the significantly higher amounts of the pro-inflammatory cytokines IL-1\u3b2 and IL-18 being produced by monocytes. In MCI, the expression of NLRP3, but not the one of PYCARD or caspase 1 was increased, indicating that functional inflammasomes are not assembled in these individuals: this was confirmed by lack of co-localization and of proinflammatory cytokines production. Conclusions The activation of at least two different inflammasome complexes explains AD-associated neuroinflammation. Strategies targeting inflammasome activation could be useful in the therapy of AD

B Lymphocytes in Multiple Sclerosis : Bregs and BTLA/CD272 Expressing-CD19+ Lymphocytes Modulate Disease Severity

B lymphocytes contribute to the pathogenesis of Multiple Sclerosis (MS) by secreting antibodies and producing cytokines. This latter function was analyzed in myelin olygodendrocyte protein (MOG)-stimulated CD19+B lymphocytes of 71 MS patients with different disease phenotypes and 40 age- and sex-matched healthy controls (HC). Results showed that: 1) CD19+/TNF alpha+, CD19+/IL-12+ and CD19+/IFN gamma+ lymphocytes are significantly increased in primary progressive (PP) compared to secondary progressive (SP), relapsing-remitting (RR), benign (BE) MS and HC; 2) CD19+/IL-6+ lymphocytes are significantly increased in PP, SP and RR compared to BEMS and HC; and 3) CD19+/IL-13+, CD19+/IL-10+, and CD19+/IL-10+/TGF beta+ (Bregs) B lymphocytes are reduced overall in MS patients compared to HC. B cells expressing BTLA, a receptor whose binding to HVEM inhibits TcR-initiated cytokine production, as well as CD19+/ BTLA+/IL-10+ cells were also significantly overall reduced in MS patients compared to HC. Analyses performed in RRMS showed that fingolimod-induced disease remission is associated with a significant increase in Bregs, CD19+/BTLA+, and CD19+/BTLA+/IL-10+ B lymphocytes. B lymphocytes participate to the pathogenesis of MS via the secretion of functionally-diverse cytokines that might play a role in determining disease phenotypes. The impairment of Bregs and CD19+/BTLA+ cells, in particular, could play an important pathogenic role in MS

TH17-driven inflammation is present in all clinical forms of multiple sclerosis; disease quiescence is associated with gata3-expressing cells

Multiple Sclerosis (MS) presents in a variety of clinical forms associated with a diverse grade of neurological impairment, different prognosis and, possibly, multiple pathogenic mechanisms. Thus, whereas relapsing-remitting (RR) MS appears to be largely driven by inflammatory processes, neurodegeneration, partially independent from inflammation, drives primary progressive (PP) and secondary progressive (SP) MS. An extensive analysis of neuroinflammation in the different forms of MS was performed by evaluating immunophenotypic and functional parameters in MBP-stimulated T lymphocytes of 103 MS patients (26 benign (BE) MS, 30 RRMS, 33 SPMS and 14 PPMS) and 40 healthy controls (HC). Results showed that: i) IL-17-producing and RORC/\u3b3t-expressing CD4+ T cells (TH17 lymphocytes), as well as IL-6 expressing CD14+ cell were augmented in all patients; ii) IL-22-expressing cells were increased in all forms of MS with the exception of PPMS; iii) TGF-\u3b2-expressing B cells were increased only in RRMS; and iv) GATA3-, NFATc-1, IL-13-, and IL-25-expressing cells (TH2 lymphocytes) were augmented in RRMS and BEMS patients alone. Data herein indicate a pivotal pathogenic role of TH17-driven inflammation in all clinical forms of MS and suggest that control over disease (RRMS and BEMS) is associated not with lack of inflammation per se, but rather with the activation of immune-mediated anti-inflammatory mechanisms. These results could help the design of novel diagnostic and therapeutic approaches

Determinants of disability in multiple sclerosis: an immunological and MRI study

Multiple sclerosis (MS) is characterized by a wide interpatient clinical variability and available biomarkers of disease severity still have suboptimal reliability. We aimed to assess immunological and MRI-derived measures of brain tissue damage in patients with different motor impairment degrees, for in vivo investigating the pathogenesis of MS-related disability. Twenty-two benign (B), 26 secondary progressive (SP), and 11 early, nondisabled relapsing-remitting (RR) MS patients and 37 healthy controls (HC) underwent conventional and diffusion tensor brain MRI and, as regards MS patients, immunophenotypic and functional analysis of stimulated peripheral blood mononuclear cells (PBMC). Corticospinal tract (CST) fractional anisotropy and grey matter volume were lower and CST diffusivity was higher in SPMS compared to RRMS and BMS patients. CD14+IL6+ and CD4+IL25+ cell percentages were higher in BMS than in SPMS patients. A multivariable model having EDSS as the dependent variable retained the following independent predictors: grey matter volume, CD14+IL6+ and CD4+IL25+ cell percentages. In patients without motor impairment after long-lasting MS, the grey matter and CST damage degree seem to remain as low as in the earlier disease stages and an immunological pattern suggestive of balanced pro- and anti-inflammatory activity is observed. MRI-derived and immunological measures might be used as complementary biomarkers of MS severity

Transcriptomic analysis of nickel exposure in Sphingobium sp. ba1 cells using RNA-seq

Nickel acts as cofactor for a number of enzymes of many bacteria species. Its homeostasis is ensured by proteins working as ion efflux or accumulation systems. These mechanisms are also generally adopted to counteract life-threatening high extra-cellular Ni2+concentrations. Little is known regarding nickel tolerance in the genus Sphingobium. We studied the response of the novel Sphingobium sp. ba1 strain, able to adapt to high Ni2+concentrations. Differential gene expression in cells cultured in 10 mM Ni2+, investigated by RNA-seq analysis, identified 118 differentially expressed genes. Among the 90 up-regulated genes, a cluster including genes coding for nickel and other metal ion efflux systems (similar to either cnrCBA, nccCBA or cznABC) and for a NreB-like permease was found. Comparative analyses among thirty genomes of Sphingobium species show that this cluster is conserved only in two cases, while in the other genomes it is partially present or even absent. The differential expression of genes encoding proteins which could also work as Ni2+-accumulators (HupE/UreJ-like protein, NreA and components of TonB-associated transport and copper-homeostasis systems) was also detected. The identification of Sphingobium sp. ba1 strain adaptive mechanisms to nickel ions, can foster its possible use for biodegradation of poly-aromatic compounds in metal-rich environments