Massimo Sanchez

Probiotics Ameliorate Recurrent Th1-Mediated Murine Colitis by Inducing IL-10 and IL-10-Dependent TGF-β-Bearing Regulatory Cells

Recent studies of murine models of mucosal inflammation suggest that, whereas some kinds of bacterial microflora are inducers of disease, others, known as probiotics, prevent disease. In the present study, we analyzed the regulatory cytokine and cell response to probiotic (VSL#3) administration in the context of the Th1 T cell colitis induced by trinitrobenzene sulfonic acid treatment of SJL/J mice. Daily administration of probiotics for 3 wk to mice during a remission period between a first and second course of colitis induced by trinitrobenzene sulfonic acid, resulted in a milder form of recurrent colitis than observed in mice administered PBS during this same period. This protective effect was attributable to effects on the lamina propria mononuclear cell (LPMC) population, because it could be transferred by LPMC from probiotic-treated mice to naive mice. Probiotic administration was associated with an early increase in the production of IL-10 and an increased number of regulatory CD4+ T cells bearing surface TGF-β in the form of latency-associated protein (LAP) (LAP+ T cells). The latter were dependent on the IL-10 production because administration of anti-IL-10R mAb blocked their appearance. Finally, the LAP+ T cells were essential to the protective effect of probiotics because administration of anti-IL-10R or anti-TGF-β at the initiation of recurrent colitis induction or depletion of LAP+ T cells from LPMC abolished the latter’s capacity to transfer protection to naive recipients. These studies show that probiotic (VSL#3) administration during a remission period ameliorates the severity of recurrent colitis by inducing an immunoregulatory response involving TGF-β-bearing regulatory cells.

Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

GATA-1 as a Regulator of Mast Cell Differentiation Revealed by the Phenotype of the GATA-1low Mouse Mutant

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neoΔHS or GATA-1low mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue+ mast cells and apoptotic metachromatic− mast cell precursors in connective tissues and peritoneal lavage and numerous (60–70% of all the progenitors) “unique” trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.

Protein Export Marks the Early Phase of Gametocytogenesis of the Human Malaria Parasite Plasmodium falciparum

Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent development of the elongated gametocyte. We developed a protocol to obtain for the first time highly purified preparations of early gametocytes using a transgenic line expressing a green fluorescent protein from the onset of gametocytogenesis. We determined the cellular proteome (1427 proteins) of this parasite stage by high accuracy tandem mass spectrometry and newly determined the proteomes of asexual trophozoites and mature gametocytes, identifying altogether 1090 previously undetected parasite proteins. Quantitative label-free comparative proteomics analysis determined enriched protein clusters for the three parasite developmental stages. Gene set enrichment analysis on the 251 proteins enriched in the early gametocyte proteome revealed that proteins putatively exported and involved in erythrocyte remodeling are the most overrepresented protein set in these stages. One-tenth of the early gametocyte-enriched proteome is constituted of putatively exported proteins, here named PfGEXPs (P. falciparum gametocyte-exported proteins). N-terminal processing and N-acetylation at a conserved leucine residue within the Plasmodium export element pentamotif were detected by mass spectrometry for three such proteins in the early but not in the mature gametocyte sample, further supporting a specific role in protein export in early gametocytogenesis. Previous reports and results of our experiments confirm that the three proteins are indeed exported in the erythrocyte cytoplasm. This work indicates that protein export profoundly marks early sexual differentiation in P. falciparum, probably contributing to host cell remodeling in this phase of the life cycle, and that gametocyte-enriched molecules are recruited to modulate this process in gametocytogenesis.

Estrogen receptor profiles in human peripheral blood lymphocytes

Estrogens are well-known regulators of the immune responses. Most of their effects are mediated by two receptors: estrogen receptor (ER)alpha and ERbeta. Up to date the presence of intracellular ER in human immune cells represents a controversial issue, while their surface membrane expression has scarcely been explored. In this study we investigated the intracellular and cell surface expression of ERalpha and ERbeta in human peripheral blood lymphocytes (PBL) by flow and static cytometry as well as by Western Blot. To this aim we used five different commercial antibodies recognizing distinct ER epitopes. We observed that CD4(+) and CD8(+) T lymphocytes, B lymphocytes and NK cells contain intracellular ERalpha and ERbeta, being the ERalpha46 isoform the most represented ER. However, significant differences could be observed among the antibodies studied in terms of immunoreactivity and specificity. Importantly, we also found a cell surface expression of ERalpha46 isoform. We also observed that a membrane-impermeant form of E2 induced a rapid phosphorylation of extracellular signal-regulated kinase (ERK), a significant proliferation of T lymphocytes, and IFN-gamma production by NK cells, thus suggesting the expression of a functional mERalpha. In conclusion our data could provide new insights as concerns the estrogen-related mechanisms of immune system modulation. They also suggest the need for a reappraisal of the experimental conditions for the characterization of the ER expression.

T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy

Autophagy, the cytoprotection mechanism that takes place under metabolic impairment, has been implicated in the pathogenesis of autoimmunity. Here, we investigated the spontaneous and induced autophagic behavior of T lymphocytes from patients with systemic lupus erythematosus (SLE) compared with that of T lymphocytes from healthy donors by measuring the autophagy marker microtubule‐associated protein 1 light chain 3 (LC3)‐II. No significant differences in spontaneous autophagy were found between T lymphocytes from patients with SLE and from healthy donors, apart from CD4+ naive T cells from patients with SLE in which constitutively higher levels of autophagy (P < 0.001) were detected. At variance, whereas treatment of T lymphocytes from healthy donors with serum IgG from patients with SLE resulted in a 2‐fold increase in LC3‐II levels (P<0.001), T lymphocytes from SLE patients were resistant to autophagic induction and also displayed an up‐regulation of genes negatively regulating autophagy, e.g., α‐synuclein. These findings could open new perspectives in the search for pathogenetic determinants of SLE progression and in the development of therapeutic strategies aimed to recover T‐cell compartment homeostasis by restoring autophagic susceptibility.—Alessandri, C., Barbati, C., Vacirca, D., Piscopo, P., Confaloni, A., Sanchez, M., Maselli, A., Colasanti, T., Conti, F., Truglia, S., Perl, A., Valesini, G., Malorni, W., Ortona, E., Pierdominici, M. T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy. FASEB J. 26, 4722–4732 (2012). www.fasebj.org

Induction of macrophage-derived chemokine/CCL22 expression in experimental autoimmune encephalomyelitis and cultured microglia: implications for disease regulation

Macrophage-derived chemokine (MDC/CCL22) and its receptor CCR4 have been implicated in chronic inflammatory processes and in the homing of monocytes, Th2 cells and regulatory T-cell subsets. Here, we demonstrate that MDC and CCR4 mRNAs are expressed in the central nervous system (CNS) of mice developing relapsing-remitting and chronic-relapsing forms of experimental autoimmune encephalomyelitis (EAE). By immunohistochemistry, we show that MDC is produced by CNS-infiltrating leukocytes and intraparenchymal microglia, whereas CCR4 is expressed on some invading leukocytes. Upon in vitro activation, mouse microglia express MDC transcripts and secrete bioactive MDC that induces chemotaxis of Th2, but not Th1 cells. We suggest that MDC produced by microglia could regulate Th1-mediated CNS inflammation by facilitating the homing of Th2 and, possibly, regulatory T cells into the lesion site.

A Transient Breach in the Epithelial Barrier Leads to Regulatory T-Cell Generation and Resistance to Experimental Colitis

BACKGROUND & AIMS Previous studies have indicated that a defective epithelial barrier leads to inflammation of the underlying lamina propria. Nevertheless, it is likely that physiologic breaks in the barrier must occur for homeostatic regulatory T cells to develop. We determined the effect of agents that disrupt epithelial tight junctions (ethanol and AT1002, a Vibrio cholerae zonula occludens toxin hexapeptide) on regulatory T-cell induction and resistance to induction of colitis by trinitrobenzene sulfonic acid (TNBS). METHODS The effects of ethanol and AT1002 on colon immune function were evaluated by their capacity to induce direct phenotypic or functional changes in effector and regulatory cell populations and their indirect effect on the development of TNBS-induced colitis. The basis of regulatory cell development was evaluated with in vitro studies of isolated dendritic cell populations. The role of innate immunity was evaluated by in vivo gene silencing studies utilizing Toll-like receptor (TLR)-2-specific small interfering RNA (siRNA). RESULTS Both ethanol and AT1002 induced persistent latency-associated peptide-positive CD4(+) regulatory T cells that, as shown in adoptive transfer studies, render mice resistant to the induction of TNBS colitis. The development of these cells requires the presence of an intact microflora and the activity of CD11c(+) dendritic cells. Their induction is also influenced by innate immune factors operating through TLR-2, because attenuation of TLR-2 signaling by in vivo TLR-2 siRNA administration prevents their development. CONCLUSIONS A mild and/or transient breach in epithelial barrier function leads to dominant regulatory T-cell responses that protect the mucosa from inflammation.

Mitochondria hyperpolarization is an early event in oxidized low‐density lipoprotein‐induced apoptosis in Caco‐2 intestinal cells

We investigated the mechanisms underlying the pro‐apoptotic activity exerted by oxidized low‐density lipoproteins (oxLDL) in Caco‐2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that: (i) oxLDL induced mitochondrial‐mediated apoptosis by provoking first an increase in mitochondrial membrane potential, followed, later, by the typical apoptosis‐associated depolarization (type II apoptosis); accordingly, (ii) caspase‐9 inhibition significantly hindered apoptosis while caspase‐8 inhibition did not; and finally (iii) dietary phenolic antioxidizing compounds exerted a significant protective antiapoptotic activity. These results point to mitochondrial hyperpolarization as ‘sensitizing feature’ in apoptotic proneness of Caco‐2 intestinal cells to oxLDL exposure.

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP‐mediated pathways. In this study, we investigated whether n‐3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2‐day‐old rats, which were then killed after 3 days post‐injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin‐specific mRNAs was analyzed by real‐time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA‐ and EPA‐treated animals, but the effect was more pronounced in EPA‐treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA‐treated rats. A further indicator of accelerated myelination was the increase in 2′‐3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) protein levels. In EPA‐treated rats, the increased expression of myelin genes coincided with a decrease of cAMP‐response element‐binding protein (CREB)‐DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA‐treated rats was less pronounced than in controls. The down‐regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n‐3 PUFA beneficial effects on several demyelinating diseases.