CD40 signaling-mediated delay in terminal differentiation of B cells enables alternate fate choices during early divisions.

Memory B cells and differentiated plasma cells combine to confer sustained humoral immunity. Nonetheless, we are yet to understand how B cells decide between these fates. Although pan-T cell help augments plasma cell differentiation, signaling via CD40 alone is considered to be inhibitory. Here, we examine the capacity of CD40 signaling to interfere with lipopolysaccharide-induced differentiation. Whereas lipopolysaccharide stimulation yielded only short-lived plasmablasts, co-stimulation of CD40 enhanced activation, proliferation, survival, and isotype-switching, leading to alternate fate choices such as germinal center and memory B cells during early divisions. Contrary to the notion that CD40 signaling simply arrests differentiation, the survivors, at later time points, developed into long-lived mature plasma cells, after progressively losing their ability to get restimulated. Counterintuitively, as constitutive lipopolysaccharide stimulation itself hampered differentiation, we identified that the proliferation potential of cells acted alongside CD40 signaling. Accordingly, we propose a bi-layered regulation of differentiation - CD40 signaling and proliferation potential of cells independently inhibit the commitment to and maturation of differentiation, respectively. Elucidating such cell fate decision mechanisms will aid in better vaccine design and disease management.

Homotypic aggregates contribute to heterogeneity in B cell fates due to an intrinsic gradient of stimulant exposure.

Monocultures of several cell types result in the formation of robust clusters called homotypic aggregates (HAs). How this physical aggregation affects cell fates in immune cell cultures, is poorly understood. We studied anti-CD40-stimulated primary B cell cultures, where cells assembled into large three-dimensional LFA1-driven HAs by 72 h. The dense packing in these aggregates restricts the infiltration of stimulants, such as antibodies, to cells inside the clusters. This creates a concentration gradient of stimulant availability across the cross-section of HAs. We describe a method to retain this positional information even after the disruption of HAs, for analysis by flow cytometry. Comparison of stage-specific cell-surface markers showed that the extent of stimulant-binding affected multiple fates non-uniformly. While germinal center and lineage markers were moderately upregulated, immunoglobulins and markers associated with memory were more than doubled in the peripheral cells binding more anti-CD40. These cells also experienced a strong repression of the plasma cell regulator Prdm1 and an upregulation of the oncogene Myc. Thus, cells at different locations in HAs are subjected to unequal doses of stimulants, leading to a hitherto unreported source of heterogeneity in cell fates. These findings can be extrapolated to understand the dose-dependent effects of stimulants in other three-dimensional cell clusters.

Learning agents in Black-Scholes financial markets.

Black-Scholes (BS) is a remarkable quotation model for European option pricing in financial markets. Option prices are calculated using an analytical formula whose main inputs are strike (at which price to exercise) and volatility. The BS framework assumes that volatility remains constant across all strikes; however, in practice, it varies. How do traders come to learn these parameters? We introduce natural agent-based models, in which traders update their beliefs about the true implied volatility based on the opinions of other agents. We prove exponentially fast convergence of these opinion dynamics, using techniques from control theory and leader-follower models, thus providing a resolution between theory and market practices. We allow for two different models, one with feedback and one with an unknown leader.

CD40-miRNA axis controls prospective cell fate determinants during B cell differentiation.

Immunological memory is a critical characteristic of a successful long-term adaptive immune response. During the initial phases of antigen:B lymphocyte interactions, B cells participate in the germinal center reaction, in which T-B cell interactions take place. CD154 on T cells acts as a ligand that binds to the CD40 receptor on B cells and facilitates the differentiation of B cells to memory B cells. However, cell fate determinants controlled by CD40 signal for cellular differentiation are unclear. In this study, we explored miRNA and miRNA-targets as cell fate determinants in CD40 signaled B cells. We selected candidate miRNAs based on their involvement in the regulation of B cell development, activation, and differentiation. We found that CD40 signal reduced transcript levels of miR150-5p, 17-5p, 146a-5p, 26a-5p and increased levels of miR292a-5p. Gene set enrichment analyses of previously submitted microarray data revealed accordant changes in levels of gene targets of these miRNA. Gene ontology analysis of miRNA-targets showed enrichment of genes participating in pathways such as DNA damage response, RNA/protein metabolism, and cell cycle regulation. Subsequently, studies on candidate miRNA-targets showed a CD40 signal driven differential regulation of Ccnd2, Pten, Traf6, c-Myb, and Btla. Further, 'gain of function' studies using mimics of the downregulated miRNAs, confirmed a predicted reduction in miRNA responsive targets; such as reduction of Ccnd2 levels in mimic treated groups of miR146a-5p, 26a-5p, and 17-5p. In conclusion, our study reveals that CD40 signal modulates levels of selected miRNAs as well as their cognate targets, whose enriched participation in diverse processes may help delineate downstream cell fate decisions.

Leishmania donovani Aurora kinase: A promising therapeutic target against visceral leishmaniasis.

BACKGROUND: Aurora kinases are key mitotic kinases executing multiple aspects of eukaryotic cell-division. The apicomplexan homologs being essential for survival, suggest that the Leishmania homolog, annotated LdAIRK, may be equally important. METHODS: Bioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy. RESULTS: Ldairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12µM and 82.9pmoles·min(-1)mg(-1) respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK-/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis. CONCLUSION: We identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability. GENERAL SIGNIFICANCE: Human homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.

Constitutive CD40 Signaling Calibrates Differentiation Outcomes in Responding B Cells via Multiple Molecular Pathways.

CD40 signaling during B cell activation is known to inhibit terminal differentiation and promote memory generation. Blimp-1 is essential for efficient plasma cell (PC) generation, and although CD40 signaling is known to inhibit Blimp-1 induction during B cell activation, the mechanisms involved have been unclear. We report that CD40 signaling induces miR-125b that targets Blimp-1 transcripts, and increases amounts of the ubiquitin ligase Hrd1 that targets BLIMP-1 protein for proteasomal degradation. CD40 signaling also inhibits the early unfolded protein response (UPR) of activated B cells that precedes the induction of terminal differentiation, and Hrd1 feeds into this pathway by targeting the core UPR component IRE-1α. Strikingly, CD40 signaling in the absence of BCR- or TLR-ligation also repressed Blimp-1 transcripts, suggesting that noncognate ligation of CD40 via T-B interactions may repress Blimp-1 in vivo. In support of this, we find that naive B cells purified from CD40-CD154 interaction-deficient mice express higher amounts of Blimp-1 and lower amounts of microRNAs and Hrd1. Higher basal amounts of Blimp-1 in naive CD40(-/-) B cells correlate with an increased tendency of the cells to undergo terminal differentiation upon LPS stimulation. Conversely, a 24-h exposure to CD40 ligation during LPS stimulation of wild-type B cells is sufficient to inhibit PC generation. The data show that CD40-mediated inhibition of PC generation is via engagement of multiple pathways that involve repression of Blimp-1 and inhibition of the UPR that prepares cells to become professional secretors. They also show that constitutive CD40 signaling in vivo involving bystander T-B interactions can calibrate B cell differentiation outcomes.

CD40 signaling drives B lymphocytes into an intermediate memory-like state, poised between naïve and plasma cells.

Immunological memory comprising of antigen-specific B and T cells contributes to the acquisition of long-term resistance to pathogens. Interactions between CD40 on B cells and CD40L on T cells are responsible for several aspects of acquired immune responses including generation of memory B cells. In order to gain insights into events leading to memory B cell formation, we analyzed the genome-wide expression profile of murine naive B cells stimulated in the presence of anti-CD40. We have identified over 8,000 genes whose expression is altered minimally 1.5-fold at least at one time point over a 3-day time course. The array analysis indicates that changes in expression level of maximum number of these genes occur within 24 h of anti-CD40 treatment. In parallel, we have studied the events following CD40 ligation by examining the expression of known regulators of naive B cell to plasma cell transition, including Pax5 and BLIMP1. The expression profile of these regulatory genes indicates firstly, that CD40 signaling activates naïve B cells to a phenotype that is intermediate between the naive and plasma cell stages of the B cell differentiation. Secondly, the major known regulator of plasma cell differentiation, BLIMP1, gets irreversibly downregulated upon anti-CD40 treatment. Additionally, our data reveal that CD40 signaling mediated BLIMP1 downregulation occurs by non-Pax5/non-Bcl6 dependent mechanisms, indicating novel mechanisms at work that add to the complexity of understanding of B cell master regulatory molecules like BLIMP1 and Pax5.

Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia.

Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-α, IL-1ß and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.

Evolutionary and functional insights into Leishmania META1: evidence for lateral gene transfer and a role for META1 in secretion.

BACKGROUND: Leishmania META1 has for long been a candidate molecule for involvement in virulence: META1 transcript and protein are up-regulated in metacyclic Leishmania. Yet, how META1 contributes to virulence remains unclear. We sought insights into the possible functions of META1 by studying its evolutionary origins. RESULTS: Using multiple criteria including sequence similarity, nucleotide composition, phylogenetic analysis and selection pressure on gene sequence, we present evidence that META1 originated in trypanosomatids as a result of a lateral gene transfer of a bacterial heat-inducible protein, HslJ. Furthermore, within the Leishmania genome, META1 sequence is under negative selection pressure against change/substitution. Using homology modeling of Leishmania META1 based on solved NMR structure of HslJ, we show that META1 and HslJ share a similar structural fold. The best hit for other proteins with similar fold is MxiM, a protein involved in the type III secretion system in Shigella. The striking structural similarity shared by META1, HslJ and MxiM suggests a possibility of shared functions. Upon structural superposition with MxiM, we have observed a putative hydrophobic cavity in META1. Mutagenesis of select hydrophobic residues in this cavity affects the secretion of the secreted acid phosphatase (SAP), indicating META1's involvement in secretory processes in Leishmania. CONCLUSIONS: Overall, this work uses an evolutionary biology approach, 3D-modeling and site-directed mutagenesis to arrive at new insights into functions of Leishmania META1.

Inhibition of terminal differentiation of B cells mediated by CD27 and CD40 involves signaling through JNK.

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream events common to both receptors.

A superantigen interacts with leishmanial infection in antigen-presenting cells to regulate cytokine commitment of responding CD4 T cells.

Germ-line retroviral insertions in vertebrate genomes are implicated in the modulation of host immune responses. We demonstrate that CBA/J mice, which carry the proviral integrants mammary tumor virus locus 6 (Mtv6) and mammary tumor virus locus 7 (Mtv7), are less resistant to infection with the protozoan pathogen Leishmania major compared with closely related but Mtv6-negative and Mtv7-negative CBA/CaJ mice. Although both strains generated comparable L. major-specific CD4 T cell frequencies, T cells from CBA/J mice made much less interferon γ (IFN-γ). L. major-infected CBA/CaJ dendritic cells primed L. major-specific and allospecific IFN-γ-producing CD4 T cells better in vivo and in vitro, respectively, than CBA/J dendritic cells did. L. major susceptibility appeared to be associated with Mtv7, and v-Sag-7 superantigen expression and L. major infection together reduced the ability of an antigen-presenting cell line to prime alloresponder CD4 T cells for IFN-γ commitment. These data show that an endogenous superantigen can interact with L. major infection to alter antigen-presenting cell properties and modulate T cell cytokine commitment, with implications for human susceptibility to infectious diseases.

Development of a real-time polymerase chain reaction assay for the quantification of Leishmania species and the monitoring of systemic distribution of the pathogen.

We have developed a highly accurate and sensitive real-time polymerase chain reaction (PCR) assay to detect and quantify Leishmania parasites. The assay targets GP63, a highly conserved gene in Leishmania. We demonstrate that, with a single assay, we are able to detect and quantify several species of Leishmania. Our assay system detects Leishmania donovani and Leishmania major down to 0.1 parasite. The dynamic range of the assay extends over 6 log cycles of target, with an average correlation coefficient >0.988. In addition, we utilize a simple approach to distinguish between Leishmania species causing diverse spectra of disease. We have also used this assay to follow the course of cutaneous disease in CBA/CaJ mice, known to be resistant to L. major. The assay is sensitive enough to quantify parasite load in the absence of overt lesions and reveals a systemic distribution of Leishmania, which has implications for our understanding of the disease.

Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides.

We demonstrate that antimicrobial peptides induce an autophagic cell death in the protozoan pathogen, Leishmania donovani. In our study, three antimicrobial peptides, Indolicidin, and two peptides derived from Seminalplasmin exhibit antileishmanial activity with a 50% lethal dose of 3.5 x 10(-5), 3.8 x 10(-4) and 1.7 x 10(-8) microM, respectively. The action of these antimicrobial peptides on the Leishmania cell involves ionic interactions, which are modulated by lipophosphoglycan on the parasite's surface. Peptide treatment caused dissipation of membrane potential and equilibration of intracellular pH with extracellular environment. However, there was no release of intracellular GFP molecules upon peptide treatment of a GFP expressing Leishmania clone. Transmission electron microscopic studies show extensive intracellular damage including cytoplasmic vacuolization and degeneration of cellular organization without disruption of the plasma membrane. These peptides induce cell death via a non-apoptotic process as shown by lack of nuclear fragmentation or DNA laddering and independent of caspase-like activity. Instead, Monodansylcadaverine (MDC), a biochemical marker of autophagy specifically labels the vacuoles induced by peptides. Collectively, these results indicate that in addition to their effects on the leishmanial membrane, these antimicrobial peptides induce pathway(s) for autophagic cell death in L. donovani.