Differential Regulation of Innate and Learned Behavior by Creb1/Crh-1 in Caenorhabditis elegans.

Memory formation is crucial for the survival of animals. Here, we study the effect of different crh-1 [Caenorhabditis elegans homolog of mammalian cAMP response element binding protein 1 (CREB1)] isoforms on the ability of C. elegans to form long-term memory (LTM). Null mutants in creb1/crh-1 are defective in LTM formation across phyla. We show that a specific isoform of CREB1/CRH-1, CRH-1e, is primarily responsible for memory related functions of the transcription factor in C. elegans Silencing of CRH-1e-expressing neurons during training for LTM formation abolishes the LTM of the animal. Further, CRH-1e expression in RIM neurons is sufficient to rescue LTM defects of creb1/crh-1-null mutants. We go on to show that apart from being LTM defective, creb1/crh-1-null animals show defects in innate chemotaxis behavior. We further characterize the amino acids K247 and K266 as responsible for the LTM related functions of CREB1/CRH-1 while being dispensable for its innate chemotaxis behavior. These findings provide insight into the spatial and temporal workings of a crucial transcription factor that can be further exploited to find CREB1 targets involved in the process of memory formation.SIGNIFICANCE STATEMENT This study elucidates the role of a specific isoform of CREB1/CRH-1, CRH-1e, in Caenorhabditis elegans memory formation and chemosensation. Removal of this single isoform of creb1/crh-1 shows defects in long-term memory formation in the animal and expression of CREB1/CRH-1e in a single pair of neurons is sufficient to rescue the memory defects seen in the mutant animals. We further show that two specific amino acids of CRH-1 are required for the process of memory formation in the animal.

FLP-18 Functions through the G-Protein-Coupled Receptors NPR-1 and NPR-4 to Modulate Reversal Length in Caenorhabditis elegans.

Animal behavior is critically dependent on the activity of neuropeptides. Reversals, one of the most conspicuous behaviors in Caenorhabditis elegans, plays an important role in determining the navigation strategy of the animal. Our experiments on hermaphrodite C. elegans show the involvement of a neuropeptide FLP-18 in modulating reversal length in these hermaphrodites. We show that FLP-18 controls the reversal length by regulating the activity of AVA interneurons through the G-protein-coupled neuropeptide receptors, NPR-4 and NPR-1. We go on to show that the site of action of these receptors is the AVA interneuron for NPR-4 and the ASE sensory neurons for NPR-1. We further show that mutants in the neuropeptide, flp-18, and its receptors show increased reversal lengths. Consistent with the behavioral data, calcium levels in the AVA neuron of freely reversing C. elegans were significantly higher and persisted for longer durations in flp-18, npr-1, npr-4, and npr-1 npr-4 genetic backgrounds compared with wild-type control animals. Finally, we show that increasing FLP-18 levels through genetic and physiological manipulations causes shorter reversal lengths. Together, our analysis suggests that the FLP-18/NPR-1/NPR-4 signaling is a pivotal point in the regulation of reversal length under varied genetic and environmental conditions.SIGNIFICANCE STATEMENT In this study, we elucidate the circuit and molecular machinery required for normal reversal behavior in hermaphrodite Caenorhabditis elegans We delineate the circuit and the neuropeptide receptors required for maintaining reversal length in C. elegans Our work sheds light on the importance of a single neuropeptide, FLP-18, and how change in levels in this one peptide could allow the animal to change the length of its reversal, thereby modulating how the C. elegans explores its environment. We also go on to show that FLP-18 functions to maintain reversal length through the neuropeptide receptors NPR-4 and NPR-1. Our study will allow for a better understanding of the complete repertoire of behaviors shown by freely moving animals as they explore their environment.

Mycobacterium indicus pranii mediates macrophage activation through TLR2 and NOD2 in a MyD88 dependent manner.

Mycobacterium indicus pranii (MIP) is a non-pathogenic strain of mycobacterium and has been used as a vaccine against tuberculosis and leprosy. Here, we investigated the role of different pattern recognition receptors in the recognition of heat-killed MIP by macrophages. Treatment of macrophages with MIP caused upregulation of pro-inflammatory cytokines (like TNFα and IL-1ß) which was mediated through both TLR2 and NOD2, as revealed by our knockdown and/or knockout studies. Mechanistically, MIP-induced macrophage activation was shown to result in NF-κB activation and drastically abrogated by MyD88 deficiency, suggesting its regulation via an MyD88-dependent, NF-κB pathway. Interestingly, the IFN-inducible cytokine, CXCL10, which is known target of the TRIF-dependent TLR pathway was found to be upregulated in response to MIP but, in an MyD88-dependent manner. Collectively, these results demonstrate macrophages to recognize and respond to MIP through a TLR2, NOD2 and an MyD88-dependent pathway. However, further studies should clarify whether additional TLR-dependent or -independent pathways also exist in regulating the full spectrum of MIP action on macrophage activation.

Mycobacterium indicus pranii downregulates MMP-9 and iNOS through COX-2 dependent and TNF-α independent pathway in mouse peritoneal macrophages in vitro.

Despite the popular belief that granulomas are innate immune mechanism to restrict mycobacterial growth, evidences suggest that granulomas facilitate growth of Mycobacterium by recruiting large numbers of uninfected macrophages to the site of infection. Matrix metalloproteinase-9 (MMP-9) has been shown to be directly involved in recruitment of macrophages at the site of infection, contributing to nascent granuloma maturation and bacterial growth. In this manuscript it is reported that heat-killed Mycobacterium indicus pranii (MIP) leads to a significant downregulation of MMP-9 in murine peritoneal macrophages in vitro. The downregulation of MMP-9 is mediated through cyclooxygenase-2 (COX-2), but independent of tumor necrosis factor-α (TNF-α). By limiting nuclear to cytoplasmic export of COX-2 and iNOS transcripts, MIP inhibits excessively-high levels of nitric oxide which can be damaging to the host during acute phases of infection. MIP has been shown to provide clinical improvement in all phases of leprosy and used for treatment of leprosy and tuberculosis.

Nod2 downregulates TLR2/1 mediated IL1ß gene expression in mouse peritoneal macrophages.

Nod2 is a cytosolic pattern recognition receptor. It has been implicated in many inflammatory conditions. Its signaling has been suggested to modulate TLR responses in a variety of ways, yet little is known about the mechanistic details of the process. We show in this study that Nod2 knockdown mouse peritoneal macrophages secrete more IL1ß than normal macrophages when stimulated with peptidoglycan (PGN). Muramyl dipeptide (MDP, a Nod2 ligand) + PGN co-stimulated macrophages have lower expression of IL1ß than PGN (TLR2/1 ligand) stimulated macrophages. MDP co-stimulation have similar effects on Pam3CSK4 (synthetic TLR2/1 ligand) mediated IL1ß expression suggesting that MDP mediated down regulating effects are receptor dependent and ligand independent. MDP mediated down regulation was specific for TLR2/1 signaling as MDP does not affect LPS (TLR4 ligand) or zymosan A (TLR2/6 ligand) mediated IL1ß expression. Mechanistically, MDP exerts its down regulating effects by lowering PGN/Pam3CSK4 mediated nuclear cRel levels. Lower nuclear cRel level were observed to be because of enhanced transporting back rather than reduced nuclear translocation of cRel in MDP + PGN stimulated macrophages. These results demonstrate that Nod2 and TLR2/1 signaling pathways are independent and do not interact at the level of MAPK or NF-κB activation.

Mycobacterium indicus pranii supernatant induces apoptotic cell death in mouse peritoneal macrophages in vitro.

Mycobacterium indicus pranii (MIP), also known as Mw, is a saprophytic, non-pathogenic strain of Mycobacterium and is commercially available as a heat-killed vaccine for leprosy and recently tuberculosis (TB) as part of MDT. In this study we provide evidence that cell-free supernatant collected from original MIP suspension induces rapid and enhanced apoptosis in mouse peritoneal macrophages in vitro. It is demonstrated that the MIP cell-free supernatant induced apoptosis is mitochondria-mediated and caspase independent and involves mitochondrial translocation of Bax and subsequent release of AIF and cytochrome c from the mitochondria. Experiments with pharmacological inhibitors suggest a possible role of PKC in mitochondria-mediated apoptosis of macrophages.

Role of prostaglandin E2 in peptidoglycan mediated iNOS expression in mouse peritoneal macrophages in vitro.

Many extracellular stimuli, e.g. microbial products, cytokines etc., result in the expression of inducible nitric oxide synthase (iNOS) in macrophages. However, it is not known whether expression of the iNOS gene in response to microbial products is a primary response of macrophages, or is the result of paracrine/autocrine signalling induced by endogenous biomolecules that are synthesised as a result of host cell-microbe interaction. In this paper we demonstrate that iNOS expression in mouse peritoneal macrophages in response to bacterial peptidoglycan (PGN) is a secondary effect requiring autocrine signalling of endogenously produced prostaglandin E2, and that PGN stimulation is mandatory, but not sufficient in itself, for induction of iNOS expression.

Protein kinase Cdelta and protein tyrosine kinase regulate peptidoglycan-induced nuclear factor-kappaB activation and inducible nitric oxide synthase expression in mouse peritoneal macrophages in vitro.

Bacteria and their ubiquitous cell wall component peptidoglycan (PGN) activate the innate immune system of the host and induce the release of inflammatory molecules. Nitric oxide (NO) is a potent molecule involved in the cytotoxic effects mediated by macrophages (MPhi) against microorganisms. This study investigates the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and nitric oxide release caused by peptidoglycan from Staphylococcus aureus in mouse peritoneal macrophages. Protein tyrosine kinase inhibitor, genestein and PKCdelta inhibitor, rottlerin attenuated the PGN-induced expression of iNOS and NO. H-7, a PKC inhibitor did not significantly affected the PGN-induced iNOS expression and NO release. NF-kappaB inhibitor, curcumin also inhibited PGN-induced NO release. Treatment of MPhi with PGN caused an increase in protein tyrosine kinase activity, expression and activation of PKCdelta, IkappaB phosphorylation and p65 (NF-kappaB) nuclear translocation. The PGN-induced IkappaB phosphorylation and p65 nuclear translocation was inhibited in macrophages pretreated with rottlerin and genestein. No paracrine or autocrine effect of TNF-alpha on PGN-induced iNOS expression and NO release was observed. These observations suggest that PGN induces enhanced expression of iNOS and NO production through activation of protein tyrosine kinases and PKCdelta, which in turn initiates NF-kappaB activation and translocation to nucleus.