Ceramide Imbalance and Impaired TLR4-Mediated Autophagy in BMDM of an ORMDL3-Overexpressing Mouse Model.

Increased orosomucoid-like 3 (ORMDL3) expression levels, due to single nucleotide polymorphisms (SNPs), have been associated with several inflammatory diseases, including asthma and inflammatory bowel diseases. ORMDL proteins inhibit serine palmitoyltransferase (SPT), the first rate-limiting enzyme in de novo sphingolipid synthesis and alter cellular calcium homeostasis. Both processes are essential for immune response. The present study addresses ORMDL3 protein involvement in macrophage physiology using an overexpressing knock-in mouse model. Ceramide content was notably different in the bone-marrow-derived macrophages (BMDM) from the transgenic mouse model compared with the wild type (WT) macrophages. Our data revealed an alteration of de novo production of sphinganine upon BMDM activation in the transgenic mouse. Gene-expression analysis showed that alteration in ORMDL3 expression levels did not affect activation or macrophage polarization. Nevertheless, we studied phagocytosis and autophagy-crucial processes that are dependent on lipid membrane composition. Phagocytosis in transgenic macrophages was not affected by ORMDL3 overexpression, but we did find a reduction in toll-like receptor 4 (TLR-4)-mediated autophagy. Both genetic and functional studies have pointed to autophagy as an essential pathway involved in inflammation. We believe that our work provides new insights into the functional link between ORMDL3 expression and inflammatory diseases.

Zip6 Transporter Is an Essential Component of the Lymphocyte Activation Machinery.

Zinc deficiency causes immune dysfunction. In T lymphocytes, hypozincemia promotes thymus atrophy, polarization imbalance, and altered cytokine production. Zinc supplementation is commonly used to boost immune function to prevent infectious diseases in at-risk populations. However, the molecular players involved in zinc homeostasis in lymphocytes are poorly understood. In this paper, we wanted to determine the identity of the transporter responsible for zinc entry into lymphocytes. First, in human Jurkat cells, we characterized the effect of zinc on proliferation and activation and found that zinc supplementation enhances activation when T lymphocytes are stimulated using anti-CD3/anti-CD28 Abs. We show that zinc entry depends on specific pathways to correctly tune the NFAT, NF-κB, and AP-1 activation cascades. Second, we used various human and murine models to characterize the zinc transporter family, Zip, during T cell activation and found that Zip6 was strongly upregulated early during activation. Therefore, we generated a Jurkat Zip6 knockout (KO) line to study how the absence of this transporter affects lymphocyte physiology. We found that although Zip6KO cells showed no altered zinc transport or proliferation under basal conditions, under activation, these KO cells showed deficient zinc transport and a drastically impaired activation program. Our work shows that zinc entry into activated lymphocytes depends on Zip6 and that this transporter is essential for the correct function of the cellular activation machinery.

The Nuclear Receptor LXR Limits Bacterial Infection of Host Macrophages through a Mechanism that Impacts Cellular NAD Metabolism.

Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of non-opsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bone-marrow-derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway.

Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21.

Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity.

Coordinated regulation of the orosomucoid-like gene family expression controls de novo ceramide synthesis in mammalian cells.

The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells.

Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion. DOI:http://dx.doi.org/10.7554/eLife.00658.001.

ORMDL3 modulates store-operated calcium entry and lymphocyte activation.

T lymphocytes rely on a Ca(2+) signal known as store-operated calcium entry (SOCE) for their activation. This Ca(2+) signal is generated by activation of a T-cell receptor, depletion of endoplasmic reticulum (ER) Ca(2+) stores and activation of Ca(2+) release-activated Ca(2+) currents (I(CRAC)). Here, we report that the ER protein orosomucoid like 3 (ORMDL3), the product of the ORMDL3 gene associated with several autoimmune and/or inflammatory diseases, negatively modulates I(CRAC), SOCE, nuclear factor of activated T cells nuclear translocation and interleukin-2 production. ORMDL3 inhibits the Ca(2+) influx mechanism at the outer mitochondrial membrane, resulting in a Ca(2+)-dependent inhibition of I(CRAC) and reduced SOCE. The effect of ORMDL3 could be mimicked by interventions that decreased mitochondrial Ca(2+) influx and reverted by buffering of cytosolic Ca(2+) or activation of mitochondrial Ca(2+) influx. In conclusion, ORMDL3 modifies key steps in the process of T-lymphocyte activation, providing a functional link between the genetic associations of the ORMDL3 gene with autoimmune and/or inflammatory diseases.

Role of type I interferons in the activation of autoreactive B cells.

Type I interferons (IFNs) are a family of cytokines involved in the defense against viral infections that play a key role in the activation of both the innate and adaptive immune system. IFNs both directly and indirectly enhance the capacity of B lymphocytes to respond to viral challenge and produce cytotoxic and neutralizing antibodies. However, prolonged type I IFN exposure is not always beneficial to the host. If not regulated properly IFN can drive autoantibody production as well as other parameters of systemic autoimmune disease. Type I IFNs impact B-cell function through a variety of mechanisms, including effects on receptor engagement, Toll-like receptor expression, cell migration, antigen presentation, cytokine responsiveness, cytokine production, survival, differentiation and class-switch recombination. Type I IFNs are also cytotoxic for a variety of cell types and thereby contribute to the accumulation of cell debris that serves as a potential source for autoantigens. Type I IFN engagement of a variety of accessory cells further promotes B-cell survival and activation, as exemplified by the capacity of type I IFNs to increase the level of B-cell survival factors, such as B lymphocyte stimulator, produced by dendritic cells. Therefore, it is not surprising that the loss of expression of the type I IFN receptor can have dramatic effects on the production of autoantibodies and on the clinical features of systemic autoimmune diseases such as systemic lupus erythematosus.

A novel Fc-engineered monoclonal antibody to CD37 with enhanced ADCC and high proapoptotic activity for treatment of B-cell malignancies.

The tetraspanin CD37 is widely expressed in B-cell malignancies and represents an attractive target for immunotherapy with mAbs. We have chimerized a high-affinity mouse Ab to CD37 and engineered the CH2 domain for improved binding to human Fcγ receptors. The resulting mAb 37.1 showed high intrinsic proapoptotic activity on malignant B cells accompanied by homotypic aggregation. Furthermore, the Ab-mediated high Ab-dependent cell-mediated cytotoxicity (ADCC) on lymphoma and primary CLL cells. mAb 37.1 strongly depleted normal B cells as well as spiked B-lymphoma cells in blood samples from healthy donors as well as malignant B cells in blood from CLL patients. In all assays, mAb 37.1 was superior to rituximab in terms of potency and maximal cell lysis. A single dose of mAb CD37.1 administered to human CD37-transgenic mice resulted in a reversible, dose-dependent reduction of peripheral B cells. In a Ramos mouse model of human B-cell lymphoma, administration of mAb 37.1 strongly suppressed tumor growth. Finally, a surrogate Fc-engineered Ab to macaque CD37, with in vitro proapoptotic and ADCC activities very similar to those of mAb 37.1, induced dose-dependent, reversible B-cell depletion in cynomolgus monkeys. In conclusion, the remarkable preclinical pharmacodynamic and antitumor effects of mAb 37.1 warrant clinical development for B-cell malignancies.

Beyond transitional selection: New roles for BLyS in peripheral tolerance.

B cell targeted therapies have enjoyed recent success in the treatment of systemic autoimmune diseases. Among these, Belimumab, which blocks the B cell survival cytokine BLyS, was recently approved for the treatment of Systemic Lupus Erythematosus. It is therefore important to consider the roles BLyS plays in B cell tolerance. Herein, we review how BLyS contributes to the negative selection of autoreactive B cell clones from the preimmune repertoire as well as its role in regulating both germinal center and extrafollicular peripheral B cell responses. We further examine the complex role of Toll-like receptors (TLRs) in humoral autoimmunity, pointing out potential crosstalk between BLyS and TLR pathways.

RAGE-independent autoreactive B cell activation in response to chromatin and HMGB1/DNA immune complexes.

Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive B cell receptor (BCR) transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1-RAGE interaction was important for this response. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1-DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE+ and RAGE(- ) AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.

Murine B cell response to TLR7 ligands depends on an IFN-beta feedback loop.

Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR(-/-) B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1(-/-) B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-beta feedback loop and constitutively low expression of TLR7 in the IFNAR1(-/-) B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.

Two distinct populations of H chain-edited B cells show differential surrogate L chain dependence.

Developing autoreactive B cells may edit (change) their specificity by secondary H or L chain gene rearrangement. Recently, using mice hemizygous for a site-directed VDJH and VJkappa transgene (tg) encoding an autoreactive Ab, we reported ongoing L chain editing not only in bone marrow cells with a pre-B/immature B cell phenotype but also in immature/transitional splenic B cells. Using the same transgenic model, we report here that editing at the H chain locus appears to occur exclusively in bone marrow cells with a pro-B phenotype. H chain editing is shown to involve VH replacement at the tg allele or VH rearrangement at the wild-type (wt) allele when the tg is inactivated by nonproductive VH replacement. VH replacement/rearrangement at the tg/wt alleles was found to entail diverse usage of VH genes. Whereas the development of edited B cells expressing the wt allele was dependent on the lambda5 component of the surrogate L chain, the development of B cells expressing the tg allele, including those with VH replacement, appeared to be lambda5 independent. We suggest that the unique CDR3 region of the tg-encoded muH chain is responsible for the lambda5 independence of tg-expressing B cells.

Antigen receptor editing in anti-DNA transitional B cells deficient for surface IgM.

In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM(-/low)). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM(-/low) anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vlambdax) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgM(low) transitional splenic B cells of wild-type mice.

The catalytic subunit of DNA-protein kinase (DNA-PKcs) is not required for Ig class-switch recombination.

The joining of DNA ends during Ig class-switch recombination (CSR) is thought to involve the same nonhomologous end-joining pathway as used in V(D)J recombination. However, we reported earlier that CSR can readily occur in Ig transgenic SCID mice lacking DNA-dependent protein kinase (DNA-PK) activity, a critical enzymatic activity for V(D)J recombination. We were thus led to question whether the catalytic subunit of DNA-PK (DNA-PKcs) is essential for CSR. To address this issue, we asked whether class switching to different Ig isotypes could occur in a line of Ig transgenic mice lacking detectable DNA-PKcs protein. The answer was affirmative. We conclude that joining of DNA ends during CSR does not require DNA-PKcs and can occur by an alternative repair pathway to that used for V(D)J recombination.

Development of functional B cells in a line of SCID mice with transgenes coding for anti-double-stranded DNA antibody.

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vkappa8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVkappa8 SCID mice) generally lack serum Ig. However, 56RVkappa8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH-/- donors. Thus, anti-dsDNA B cells that escape deletion in 56RVkappa8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.

Human peripheral CD2-/lo T cells: an extrathymic population of early differentiated, developing T cells.

We previously reported that a subset of human peripheral blood CD3+ T cells expresses low-to-null CD2 levels (CD2-/lo), produces type 2 cytokines and is inducible to differentiate to functionally mature IFN-gamma+ cells. Multiple-color immunofluorescence analysis indicated that this population, representing <0.1% of the T cells in fresh lymphocytes, contains subsets that are phenotypically immature, including CD4-CD8- and CD3+TCR- cells. Ex vivo, the CD2-/lo cells can proliferate (carboxyfluorescein diacetate succinimidyl ester analysis) independently from exogenous stimulation, respond to CD3-mediated stimulation with significantly greater proliferation than the autologous mature cells and their subsets are inducible to undergo in vitro a developmental sequence similar to that reported for the phenotypically similar thymic populations. This is especially evident for the CD4+CD8+ subset. CD2-/lo T-cell populations exhibit a TCR repertoire (Vbeta chain distribution) that is complete but different (complementarity determining region R3 analysis) from that of the autologous CD2+ T cells. These characteristics distinguish peripheral CD2-/lo T cells as possible early differentiated T cells that may undergo extrathymic maturation, and potentially contribute to maintain the peripheral naive T-cell pool. These findings define the existence of phenotypically immature T cells in the periphery. Also, given the high numbers of CD2-/lo T cells generated, upon ex vivo culture, from peripheral lymphocytes of all adult and neonatal individuals tested, they have relevance to clinical applications for immune reconstitution of T cells, as well as myeloid cells, via myeloid colony-stimulating factors and type 2 cytokines.