Federica Piancone

The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer's disease

BackgroundInterleukin-1 beta (IL-1β) and its key regulator, the inflammasome, are suspected to play a role in the neuroinflammation observed in Alzheimer’s disease (AD); no conclusive data are nevertheless available in AD patients.ResultsmRNA for inflammasome components (NLRP1, NLRP3, PYCARD, caspase 1, 5 and 8) and downstream effectors (IL-1β, 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-1β 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.ConclusionsThe activation of at least two different inflammasome complexes explains AD-associated neuroinflammation. Strategies targeting inflammasome activation could be useful in the therapy of AD.

Increased activity of Th-17 and Th-9 lymphocytes and a skewing of the post-thymic differentiation pathway are seen in Alzheimer’s disease

Inflammatory mediators are responsible for the neuroinflammation observed in Alzheimers disease (AD), a phenomenon that might be the culprit of disease or, possibly, a reaction to pathology. To better investigate inflammation in AD we performed an extensive immunophenotypic and functional analysis of amyloid-beta (Aβ) stimulated T lymphocytes in patients with a diagnosis of AD comparing data to those obtained in individuals with mild cognitive impairment (MCI) or aged-matched healthy individuals (HC). Results showed that IL-21- and IL-9-producing Aβ stimulated CD4(+) T cells, as well as IL-23- and IL-6-producing monocytes and CD4(+) T cells expressing the RORγ and NFATc1 transcriptional factors (TF), were significantly increased, whereas IL-10-producing monocytes were decreased in AD. Notably, GATA-3 TF-expressing CD4(+) T lymphocytes were significantly increased in MCI alone. Analysis of the post-thymic differentiation pathway indicated that Aβ specific naïve and central memory CD4(+) T lymphocytes were diminished whereas effector memory and terminally differentiated CD4(+) T lymphocytes were increased in AD and MCI compared to HC. Data herein indicate that cytokines (IL-21, IL-6, IL-23) and TF (RORγ) involved in the differentiation of Th-17 cells), as well as cytokines (IL-21, IL-22) generated by such cells, and IL-9, produced by Th-9 cells, are significantly increased in AD. This is accompanied by a shift of post-thymic differentiation pathways favoring the accumulation of differentiated, effector T lymphocytes. These data shed light on the nature of AD-associated neuroinflammation. A better understanding of the complexity of this phenomenon could facilitate the search for novel therapeutic strategies.

PD1 Negative and PD1 Positive CD4+ T Regulatory Cells in Mild Cognitive Impairment and Alzheimer's Disease

Regulatory T lymphocytes (Treg) play a fundamental importance in modulating the relative balance between inflammation and immune tolerance, and alterations of these cells are observed in inflammatory diseases. To better characterize the neuroinflammatory processes suggested to be associated with Alzheimers disease (AD) and to clarify the possible role of Treg cells in this process, we extensively analyzed these cells (CD4 + CD25highFoxp3+) in patients with either severe AD (n=25) or mild cognitive impairment (MCI) (n=25), comparing the results with those of two groups of healthy controls (HC) (n=55). Because the intra- or extracellular expression of programmed death receptor 1 (PD1) identifies functionally diverse subsets of Treg we also analyzed such subpopulations. Results showed that, whereas both Treg and PD1pos Treg are increased in MCI and AD patients compared to HC, PD1neg Treg, the subpopulation of Treg cells endowed with the strongest suppressive ability, are significantly augmented in MCI patients alone. In these patients amyloid-β-stimulated-T cells proliferation was reduced and Treg-mediated suppression was more efficient compared to both AD and HC. The observation that PD1neg Treg, cells are increased in MCI patients reinforces the inflammatory origin of AD and supports a possible beneficial role of these cells in MCI that is lost in patients with full-blown AD.

A complex proinflammatory role for peripheral monocytes in Alzheimer's disease

An impairment of the microglial catabolic mechanisms allows amyloid-β (Aβ) accumulation in plaques within the brain in Alzheimers disease (AD). Monocytes/macrophages (M/M) are activated in AD and migrate thorough the blood-brain barrier (BBB) trying to improve Aβ clearing. In the attempt to shed light on the role of M/M in AD, these cells were analyzed in patients with AD or mild cognitive impairment (MCI) and in age-matched healthy controls. Results obtained in Aβ42-stimulated cell cultures showed that significantly higher percentages of inflammatory M/M (CD14+ CD16-CCR2++CX3CR1low) expressing toll like receptors (TLR) 2 and 4, as well as IL-6 and CCR2, a chemokine favoring M/M migration through the BBB, are seen in AD. Confocal microscopy suggested the presence of MHC-II/Aβ42 complexes on AD M/M alone. Finally, TRL3- and TLR8-expressing and IL-23-producing M/M were increased in both AD and MCI compared to HC. These data indicate that M/M in AD are characterized by an inflammatory profile and are involved in the induction of both innate immune responses via TLR stimulation and of acquired immunity possibly secondarily to the presentation of Aβ peptides in an MHC-restricted fashion. Therapeutic approaches designed to interrupt these mechanism might prove beneficial.

T helper-17 activation dominates the immunologic milieu of both amyotrophic lateral sclerosis and progressive multiple sclerosis

MS (multiple sclerosis) and ALS (amyotrophic lateral sclerosis) differ in important respects, but common pathogenic features seem to be shared in these two diseases. To shed light on such features, immunophenotypic and functional analysis were performed in peripheral monocytes and T lymphocytes of ALS and primary progressive (PP) MS patients and healthy controls (HC). Results showed that TH1-, TH17-, and IL-6-driven inflammation characterize both diseases; this is unsuccessfully hampered by TH2 activation and, possibly, BDNF secretion. Results herein clarify the pathogenic similarities between ALS and PP-MS and could be helpful for the design of novel diagnostic and therapeutic approaches to ALS.

A potential role for the PD1/PD-L1 pathway in the neuroinflammation of Alzheimer's disease

The interaction between PD1 on T lymphocytes and PD-L1 on antigen presenting cells (APC) modulates the balance between inflammation and tolerance by inducing IL-10 production and apoptosis of antigen-specific cells. We analyzed the PD1/PD-L1 pathway, annexin V (AV)-expression, and proliferation in amyloid-beta (Aβ)-stimulated PBMC of patients with Alzheimers disease (AD) (N = 35) or mild cognitive impairment (MCI) (N = 30) and of age-matched healthy controls (HC; N = 30). Results showed that PD1-expressing CD4(+) T cells, density of PD-L1 on CD14(+) APC, IL-10 production, and PD-L1-expressing/IL-10-producing CD14(+) APC were significantly reduced in AD and MCI patients compared to HC. Aβ-stimulated PD1/AV-expressing (apoptotic) CD4(+) T cells were also diminished, whereas proliferation was augmented in AD and MCI patients compared to controls. Finally, incubation of cells with PD-L1-neutralizing antibodies significantly decreased apoptosis of Aβ-specific CD4(+) T lymphocytes. An impairment of the PD-L1/PD1 pathway is present in AD and MCI. Such alteration results in reduced IL-10 production and diminished apoptosis of Aβ-specific CD4(+) T lymphocytes; these phenomena could play a role in the neuroinflammation accompanying AD.

Neuroinflammation and brain functional disconnection in Alzheimer's disease.

Neuroinflammation and brain functional disconnection result from β-amyloid (Aβ) accumulation and play fundamental roles in the pathogenesis of Alzheimer’s disease (AD). We investigated possible correlations between these two AD-associated phenomena using DTI-based tractography and immunologic analyses in people with amnestic mild cognitive impairment (aMCI) and AD. DTI-Analyses focused on corpus callosum (CC). We found that frontal CC regions were preserved with respect to the posterior ones in aMCI; in these individuals significant correlations were seen between DTI-derived metrics in frontal-parietal CC areas and Aβ42-stimulated BDNF-producing CD4+ T lymphocytes and PDL-1-expressing CD14+ cells. These associations were lost in AD where DTI data involving the same CC areas correlated instead with Aβ42-stimulated interleukin (IL)-21 producing CD4+ T lymphocytes. Higher susceptibility to PDL-1-mediated apoptosis of Aβ42-specific lymphocytes and BDNF-associated survival of existing neurons could contribute to the relative CC structure preservation seen in aMCI. These potentially protective mechanisms are lost in frank AD, when severe alterations in the CC are mirrored in peripheral blood by proinflammatory cytokines-producing T cells. Monitoring of immune cells in peripheral blood could have a prognostic value in AD.

A role for the TIM-3/GAL-9/BAT3 pathway in determining the clinical phenotype of multiple sclerosis

T‐cell immunoglobulin and mucin domain 3 (Tim‐3) ligates galectin‐9 (Gal‐9); this process, resulting in the inhibition of Th1 responses and in the apoptosis of antigen‐specific cells, is hampered by binding of the molecular adaptor human leukocyte antigen B (HLA‐B)‐associated transcript 3 (Bat3) to the intracellular tail of Tim‐3. Apoptosis of myelin basic protein (MBP)‐specific T lymphocytes correlates with reduced rates of disease progression in multiple sclerosis (MS). We extensively analyzed the Tim‐3/Gal‐9/Bat3 pathway in 87 patients with a diagnosis of stable relapsing‐remitting MS (RRMS), primary progressive MS (PPMS), or benign MS (BEMS), as well as in 40 healthy control (HC) subjects. Results showed that MBP‐specific CD4+Tim‐3+, CD4+/Gal‐9+, and CD4+/Tim‐3+/AV+ (apoptotic) T lymphocytes were augmented in the BEMS group, whereas CD4+/Bat3+ and CD8+/Bat3+ T lymphocytes were increased and CD4+/Tim‐3+/AV+ T cells were reduced in the PPMS group (>2 fold and P<0.05 in all cases). Blocking the Tim‐3/Gal‐9 interaction with specific mAb reduced T‐lymphocyte apoptosis and augmented production of IFNγ and IL‐17 in the BEMS, RRMS, and HC groups, but not in the PPMS group. The Tim‐3/Gal‐9 interaction favors apoptosis of MBP‐specific T lymphocytes in BEMS; this process is reduced in PPMS by the up‐regulation of Bat3. Therapeutic interventions aimed at silencing Bat3 could be beneficial in MS.—Saresella, M., Piancone, F., Marventano, I., La Rosa, F., Tortorella, P., Caputo, D., Rovaris, M., Clerici, M., A role for the TIM‐3/GAL‐9/BAT3 pathway in determining the clinical phenotype of multiple sclerosis. FASEB J. 28, 5000–5009 (2014). www.fasebj.org

Torque teno virus (TTV) in multiple sclerosis patients with different patterns of disease.

Torque teno virus (TTV) is highly prevalent in the general population worldwide. The relationship that TTV establishes with the central nervous system (CNS) of infected hosts is not clear but it is suspected that TTV infection of the CNS lead to increased local expression of inflammatory mediators that may play a role in the pathogenesis of multiple sclerosis (MS). The prevalence and load of TTV in serum and cerebrospinal fluid (CSF) of 207 MS patients and 93 age‐ and sex‐matched healthy controls by qPCR designed on the untranslated region were analyzed. TTV DNA was not detected in CSF, TTV prevalence in serum was similar in MS patients (76.8%) compared to healthy controls (76.3%). Sub analyses performed in MS patients stratified on the basis of clinical phenotypes indicated that TTV viremia was significantly lower in individuals with relapsing remitting compared to chronic progressive disease. Notably, viremia was increased in primary progressive, compared to secondary progressive MS patients, and in relapsing remitting MS patients during quiescent compared to relapsing phases of disease. Since TTV interacts with toll‐like receptor (TLR) stimulating the production of inflammatory cytokines, TLR9 expression were examined, showing that it was augmented on monocytes of chronic progressive MS patients, in whom higher TTV viremia was present, but this did not correlate with a distinct pattern of cytokine production. Overall these findings suggest that, although TTV infects the same proportion of MS patients and healthy controls, the levels of replication of the virus differ among patients, being correlated with the clinical phenotype of disease. J. Med. Virol. 85:2176–2183, 2013.

Immunological and clinical effect of diet modulation of the gut microbiome in multiple sclerosis patients: A pilot study

Pathogenesis of autoimmune disorders, including multiple sclerosis (MS), has been linked to an alteration of the resident microbial commensal community and of the interplay between the microbiota and the immune system. Dietary components such as fiber, acting on microbiota composition, could, in principle, result in immune modulation and, thus, could be used to obtain beneficial outcomes for patients. We verified this hypothesis in a pilot study involving two groups of clinically similar relapsing-remitting (RR) MS patients who had undergone either a high-vegetable/low-protein diet (HV/LP diet group; N = 10) or a “Western Diet” (WD group; N = 10) for at least 12 months. Gut microbiota composition, analyzed by 16 S V4 rRNA gene sequencing and immunological profiles, was examined after a minimum of 12 months of diet. Results showed that, in the HV/LP diet group compared to the WD group: (1) Lachnospiraceae family was significantly more abundant; (2) IL-17-producing T CD4+ lymphocytes (p = 0.04) and PD-1 expressing T CD4+ lymphocytes (p = 0.0004) were significantly decreased; and (3) PD-L1 expressing monocytes (p = 0.009) were significantly increased. In the HV/LP diet group, positive correlations between Lachnospiraceae and both CD14+/IL-10+ and CD14+/TGFβ+monocytes (RSp = 0.707, p = 0.05, and RSp = 0.73, p = 0.04, respectively), as well as between Lachnospiraceae and CD4+/CD25+/FoxP3+ T lymphocytes (RSp = 0.68, p = 0.02) were observed. Evaluation of clinical parameters showed that in the HV/LP diet group alone the relapse rate during the 12 months follow-up period and the Expanded Disability Status Scale score at the end of the study period were significantly reduced. Diet modulates dysbiosis and improves clinical parameters in MS patients by increasing anti-inflammatory circuits. Because Lachnospiraceae favor Treg differentiation as well as TGFβ and IL-10 production this effect could be associated with an increase of these bacteria in the microbiota.