Patricia López

Epidemiology of systemic lupus erythematosus in a northern Spanish population: gender and age influence on immunological features.

The present work was planned to research epidemiological and immunological features of systemic lupus erythematosus (SLE) in a Caucasian population from the north of Spain (Asturias). There is only one specialized immunology laboratory in this region where samples from all patients with a plausible or a firm diagnosis of SLE are referred for immunological analysis. Since 1992 we have reviewed registered data from samples submitted to the immunology laboratory with a firm, definitive diagnosis of SLE, based on the fulfillment of the American College of Rheumatology (ACR) criteria. We have constructed a database, which included 367 SLE patients. The point prevalencewas 34.12/100 000 (95% CI: 30.63-37.61/100 000), whereas the incidence rate calculated during the last five years was 2.15/100 000/year (95% CI: 1.76-2.54/100 000/year). The female/male ratio varied according to the age at diagnosis, being maximum (50: 1) between 22 and 28 years. The median age at diagnostis was significantly lower in females than in males. Immunological features also yielded sex and age peculiarities. The percentage of patients with anti-SSa antibodies yielded significant differences between males (18.6%) and females (34.6%). Anti-RNP and anti-Sm antibodies were more frequently present in childhood-onset patients, the difference with the oldest-onset group being statistically significant. Other analyses did not show significant differences, although, as a whole, we observed a trend towards a higher presence of autoantibodies related to an early disease onset.

Intestinal Dysbiosis Associated with Systemic Lupus Erythematosus

ABSTRACT Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disease in humans and is characterized by the presence of hyperactive immune cells and aberrant antibody responses to nuclear and cytoplasmic antigens, including characteristic anti–double-stranded DNA antibodies. We performed a cross-sectional study in order to determine if an SLE-associated gut dysbiosis exists in patients without active disease. A group of 20 SLE patients in remission, for which there was strict inclusion and exclusion criteria, was recruited, and we used an optimized Ion Torrent 16S rRNA gene-based analysis protocol to decipher the fecal microbial profiles of these patients and compare them with those of 20 age- and sex-matched healthy control subjects. We found diversity to be comparable based on Shannon’s index. However, we saw a significantly lower Firmicutes/Bacteroidetes ratio in SLE individuals (median ratio, 1.97) than in healthy subjects (median ratio, 4.86; P < 0.002). A lower Firmicutes/Bacteroidetes ratio in SLE individuals was corroborated by quantitative PCR analysis. Notably, a decrease of some Firmicutes families was also detected. This dysbiosis is reflected, based on in silico functional inference, in an overrepresentation of oxidative phosphorylation and glycan utilization pathways in SLE patient microbiota. IMPORTANCE Growing evidence suggests that the gut microbiota might impact symptoms and progression of some autoimmune diseases. However, how and why this microbial community influences SLE remains to be elucidated. This is the first report describing an SLE-associated intestinal dysbiosis, and it contributes to the understanding of the interplay between the intestinal microbiota and the host in autoimmune disorders. Growing evidence suggests that the gut microbiota might impact symptoms and progression of some autoimmune diseases. However, how and why this microbial community influences SLE remains to be elucidated. This is the first report describing an SLE-associated intestinal dysbiosis, and it contributes to the understanding of the interplay between the intestinal microbiota and the host in autoimmune disorders.

Enrichment of CD4+ CD25high T cell population in patients with systemic lupus erythematosus treated with glucocorticoids.

Objectives: To characterise and quantify the CD4+ CD25+ T cell population in patients with systemic lupus erythematosus (SLE) and to detect the possible influence of treatments and clinical manifestations. Methods: Characterisation of CD25low and CD25high CD4+ T cells from healthy controls and from patients with SLE was carried out using flow cytometry, analysing the expression of activation and differentiation markers. The percentage of both circulating cell subsets was determined in 56 controls and 110 unselected patients with SLE. Data were related to treatment during the past 3 months and to various clinical manifestations. Results: CD4+ CD25high lymphocytes from controls expressed low levels of CD69, CD154 or CD30, but also expressed glucocorticoid-induced tumour necrosis factor receptor, high levels of intracellular cytotoxin T lymphocyte-associated antigen 4, CD45RO and diminished amounts of CD4, all of which are phenotypic characteristics of natural regulatory T cells. CD4+ CD25low cells, on the other hand, expressed the highest levels of activation markers, indicating that they represent recently activated effector cells. Similarly, analysis of cells from patients with SLE showed the same two phenotypically distinguishable CD4+ CD25low and CD4+ CD25high populations, although both expressed slightly increased levels of activation markers. Quantitative analysis showed a considerably raised percentage of CD25low and, especially, CD25high cells in patients with SLE compared with controls. This increment was unrelated to clinical manifestations, but correlated with glucocorticoid treatment. Patients treated with glucocorticoids presented raised levels of CD25high cells, whereas untreated patients and those with anti-malarial or immunosuppressive drugs had levels similar to those in controls. Conclusions: The percentage of CD4+ CD25high cells was not altered in non-steroid-treated patients, whereas glucocorticoid treatment increased their frequency in patients with SLE.

Distinct Bifidobacterium strains drive different immune responses in vitro

In this work we evaluated the specific immune activation properties of different Bifidobacterium strains, some of the most relevant intestinal microorganisms. To this end, we examined the in vitro effect of 12 Bifidobacterium strains belonging to 4 different species, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium animalis subsp. lactis, on the maturation pattern of human monocyte-derived dendritic cells (DCs), as well as in their ability to induce cytokine secretion. In addition, we determined peripheral blood mononuclear cell (PBMC) proliferation and cytokine expression after exposure to bacterial strains. All bifidobacteria tested were able to induce full DC maturation but showed differences in the levels of cytokine production, especially IL-12, IL-10, TNFalpha and IL-1beta, suggesting that specific cytokine ratios could be used to predict the type of Th response that they may promote. In fact, analysis of cytokine production by PBMC showed that most of the tested B. animalis and B. longum strains induced the secretion of large amounts of IFNgamma and TNFalpha, in agreement with the Th1 profile suggested by DC cytokine production. Remarkably, three of four B. bifidum strains induced poor secretion of these cytokines and significant amounts of IL-17, the main product of Th17 cells, in accordance with the high IL-1beta/IL-12 ratio observed after DC stimulation. In conclusion, this work shows species and strain-specific immune effects of bifidobacteria and describes a valuable method for screening possible probiotic strains with different immunomodulatory properties. Notably, some B. bifidum strains seem to promote Th17 polarization, which could be useful for future probiotic applications.

Differential effect of IL10 and TNFα genotypes on determining susceptibility to discoid and systemic lupus erythematosus

Objective: To ascertain the possible involvement of functional interleukin 10 (IL10) and tumour necrosis α (TNFα) cytokine promoter polymorphisms on the susceptibility to discoid and systemic lupus erythematosus (DLE, SLE), and their associations with immunological features. Methods: Single nucleotide polymorphisms of the IL10 (−1082, −819, and −592) and TNFα (−308) genes were determined using allele specific probes in 248 lupus patients and 343 matched controls. To assess functional significance of genotypes, basal mRNA cytokine levels were quantified in 106 genotyped healthy controls by real time RT-PCR. Specific autoantibodies and cutaneous manifestations were analysed in SLE patients and associated with functional genotypes. Results: After analysing the distribution of IL10 and TNFα transcript levels according to promoter genotypes in healthy individuals, patients and controls were classified into functional single and combined genotypes according to the expected high or low constitutive cytokine production. High TNFα genotypes (−308AA or AG) were associated with SLE independently of IL10 alleles, whereas the risk of developing DLE and the prevalence of discoid lesion in SLE were higher in the high IL10/low TNFα producer group (−1082GG/−308GG). Cytokine interaction also influences the appearance of autoantibodies. Antibodies against Sm are prevalent among low producer patients for both cytokines, a genotype not associated with lupus incidence, whereas low IL10/high TNFα patients have the highest frequency of antibodies to SSa and SSb. Conclusions: IL10/TNFα interaction influences susceptibility to DLE and the appearance of specific autoantibodies in SLE patients, whereas high TNFα producer genotypes represent a significant risk factor for SLE.

Immune Modulation Capability of Exopolysaccharides Synthesised by Lactic Acid Bacteria and Bifidobacteria

During recent years, the exopolysaccharides (EPS) produced by some strains of lactic acid bacteria and bifidobacteria have attracted the attention of researchers, mainly due to their potential technological applications. However, more recently, it has been observed that some of these EPS present immunomodulatory properties, which suggest a potential effect on human health. Whereas EPS from lactic acid bacteria have been studied in some detail, those of bifidobacteria largely remain uncharacterized in spite of the ubiquity of EPS genes in Bifidobacterium genomes. In this review, we have analysed the data collected in the literature about the potential immune-modulating capability of EPS produced by lactic acid bacteria and bifidobacteria. From this data analysis, as well as from results obtained in our group, a hypothesis relating the physicochemical characteristics of EPS with their immune modulation capability was highlighted. We propose that EPS having negative charge and/or small size (molecular weight) are able to act as mild stimulators of immune cells, whereas those polymers non-charged and with a large size present a suppressive profile.

Immune response to Bifidobacterium bifidum strains support Treg/Th17 plasticity.

In this work we analyzed the immune activation properties of different Bifidobacterium strains in order to establish their ability as inductors of specific effector (Th) or regulatory (Treg) responses. First, we determined the cytokine pattern induced by 21 Bifidobacterium strains in peripheral blood mononuclear cells (PBMCs). Results showed that four Bifidobacterium bifidum strains showed the highest production of IL-17 as well as a poor secretion of IFNγ and TNFα, suggesting a Th17 profile whereas other Bifidobacterium strains exhibited a Th1-suggestive profile. Given the key role of Th17 subsets in mucosal defence, strains suggestive of Th17 responses and the putative Th1 Bifidobacterium breve BM12/11 were selected to stimulate dendritic cells (DC) to further determine their capability to induce the differentiation of naïve CD4+ lymphocytes toward different Th or Treg cells. All selected strains were able to induce phenotypic DC maturation, but showed differences in cytokine stimulation, DC treated with the putative Th17 strains displaying high IL-1β/IL-12 and low IL-12/IL-10 index, whereas BM12/11-DC exhibited the highest IL-12/IL-10 ratio. Differentiation of naïve lymphocytes confirmed Th1 polarization by BM12/11. Unexpectedly, any B. bifidum strain showed significant capability for Th17 generation, and they were able to generate functional Treg, thus suggesting differences between in vivo and vitro responses. In fact, activation of memory lymphocytes present in PBMCS with these bacteria, point out the presence in vivo of specific Th17 cells, supporting the plasticity of Treg/Th17 populations and the key role of commensal bacteria in mucosal tolerance and T cell reprogramming when needed.

Ranking the impact of human health disorders on gut metabolism: Systemic lupus erythematosus and obesity as study cases

Multiple factors have been shown to alter intestinal microbial diversity. It remains to be seen, however, how multiple collective pressures impact the activity in the gut environment and which, if any, is positioned as a dominant driving factor determining the final metabolic outcomes. Here, we describe the results of a metabolome-wide scan of gut microbiota in 18 subjects with systemic lupus erythematosus (SLE) and 17 healthy control subjects and demonstrate a statistically significant difference (p < 0.05) between the two groups. Healthy controls could be categorized (p < 0.05) based on their body mass index (BMI), whereas individuals with SLE could not. We discuss the prevalence of SLE compared with BMI as the dominant factor that regulates gastrointestinal microbial metabolism and provide plausible explanatory causes. Our results uncover novel perspectives with clinical relevance for human biology. In particular, we rank the importance of various pathophysiologies for gut homeostasis.

Th17 responses and natural IgM antibodies are related to gut microbiota composition in systemic lupus erythematosus patients.

Intestinal dysbiosis, characterized by a reduced Firmicutes/Bacteroidetes ratio, has been reported in systemic lupus erythematosus (SLE) patients. In this study, in vitro cultures revealed that microbiota isolated from SLE patient stool samples (SLE-M) promoted lymphocyte activation and Th17 differentiation from naïve CD4+ lymphocytes to a greater extent than healthy control-microbiota. Enrichment of SLE-M with Treg-inducing bacteria showed that a mixture of two Clostridia strains significantly reduced the Th17/Th1 balance, whereas Bifidobacterium bifidum supplementation prevented CD4+ lymphocyte over-activation, thus supporting a possible therapeutic benefit of probiotics containing Treg-inducer strains in order to restore the Treg/Th17/Th1 imbalance present in SLE. In fact, ex vivo analyses of patient samples showed enlarged Th17 and Foxp3+ IL-17+ populations, suggesting a possible Treg-Th17 trans-differentiation. Moreover, analyses of fecal microbiota revealed a negative correlation between IL-17+ populations and Firmicutes in healthy controls, whereas in SLE this phylum correlated directly with serum levels of IFNγ, a Th1 cytokine slightly reduced in patients. Finally, the frequency of Synergistetes, positively correlated with the Firmicutes/Bacteroidetes ratio in healthy controls, tended to be reduced in patients when anti-dsDNA titers were increased and showed a strong negative correlation with IL-6 serum levels and correlated positively with protective natural IgM antibodies against phosphorylcholine.

Interaction of Bifidobacterium bifidum LMG13195 with HT29 Cells Influences Regulatory-T-Cell-Associated Chemokine Receptor Expression

ABSTRACT Probiotics play an important role in the maintenance of the gastrointestinal barrier. In addition to direct effects on mucosal integrity, the interaction with the intestinal mucosa may have an active immunoregulatory effect. In the present work, we exposed HT29 intestinal epithelial cells to two Bifidobacterium species to determine their effect on gene expression profile, enterocyte monolayer integrity, and T-cell response. Bifidobacterium breve IPLA 20004 triggered a more pronounced increase in the transepithelial resistance of the enterocyte monolayer than Bifidobacterium bifidum LMG13195. The transcriptome profile of HT29 cells cultured in the presence of B. bifidum LMG13195 showed an increased expression of immune mediators and, interestingly, chemotactic molecules (CXCL10, CCL20, CXCL11 and CCL22) able to recruit lymphocytes. Since regulatory T cells (Treg cells) may express receptors for specific chemokines, we cultured peripheral blood mononuclear cells with supernatants of HT29 cells previously treated with Bifidobacterium strains and analyzed FOXP3 and CD25 Treg markers and CCR6, CXCR3, CCR4, and CCR3 expression on CD4+ lymphocytes. The proportion of CD25high FOXP3+ cells was significantly increased after culture with B. bifidum LMG13195-conditioned HT29 supernatant. Moreover, this treatment led to the largest amount of CCR6+ CXCR3− CCR4+ CCR3+ CD4+ cells expressing high levels of CD25, corresponding to the Treg population. These results suggest that soluble factors secreted after B. bifidum LMG13195 contact with intestinal epithelial cells favored the generation of CD4+ CD25high lymphocytes expressing chemokine receptor Treg markers, thus making possible their recruitment to the intestinal mucosa.