Shibnath Mazumder

Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

We had earlier shown that exposure to arsenic (0.50 μM) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca(2+)) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca(2+) homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca(2+) levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus.

Role of Calmodulin-Calmodulin Kinase II, cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.

Aeromonas hydrophila induced head kidney macrophage apoptosis in Clarias batrachus involves the activation of calpain and is caspase-3 mediated

The mechanism of macrophage cytotoxicity induced by Aeromonas hydrophila is yet unresolved. We observed A. hydrophila induces Head Kidney Macrophage (HKM) apoptosis in Clarias batrachus, as evident from Hoechst 33342 and AnnexinV-Propidium Iodide staining and presence of oligonucleosomal DNA ladder. Initiation of apoptosis required the bacteria to be alive, be actively phagocytosed into HKM and was dependent on host proteins. Elevated cytosolic calcium and consequent calpain activity that declined following pre-incubation with EGTA, verapamil and nifedipine implicates the role of calcium influx through voltage gated calcium channels and calpain in A. hydrophila-induced HKM apoptosis. Though, calpain-1 and -2 were involved, calpain-2 appeared to be more important in the process. EGTA, verapamil, nifedipine and calpain-2 inhibitor reduced caspase-3 activity and apoptosis. We conclude that A. hydrophila alters cytosolic calcium homeostasis initiating the activation of calpains, more specifically calpain-2, which leads to caspase-3 mediated HKM apoptosis in C. batrachus.

Ameliorating ER-stress attenuates Aeromonas hydrophila-induced mitochondrial dysfunctioning and caspase mediated HKM apoptosis in Clarias batrachus.

Endoplasmic reticulum (ER)-stress and unfolding protein response (UPR) has not been implied in Aeromonas hydrophila-pathogenicity. We report increased expression of the ER-stress markers: CHOP, BiP and phospho-eIF2α in A. hydrophila-infected headkidney macrophages (HKM) in Clarias batrachus. Pre-treatment with ER-stress inhibitor, 4-PBA alleviated ER-stress and HKM apoptosis suggesting ER-UPR critical for the process. The ER-Ca2+ released via inositol-triphosphate and ryanodine receptors induced calpain-2 mediated superoxide ion generation and consequent NF-κB activation. Inhibiting NF-κB activation attenuated NO production suggesting the pro-apoptotic role of NF-κB on HKM pathology. Calpain-2 activated caspase-12 to intensify the apoptotic cascade through mitochondrial-membrane potential (ψm) dissipation and caspase-9 activation. Altered mitochondrial ultra-structure consequent to ER-Ca2+ uptake via uniporters reduced ψm and released cytochrome C. Nitric oxide induced the cGMP/PKG-dependent activation of caspase-8 and truncated-Bid formation. Both the caspases converge onto caspase-3 to execute HKM apoptosis. These findings offer a possible molecular explanation for A. hydrophila pathogenicity.

Isolation and Characterization of Arsenic-Resistant Bacteria from Contaminated Water-Bodies in West Bengal, India

Bengal Basin is known for severe arsenic contamination. In the present study, we have isolated six bacteria from the arsenic contaminated surface water of Bengal Basin. 16S rDNA sequence analysis identified them as Microbacterium oleivorans, Acinetobacter soli, Acinetobacter venetianus, Acinetobacter junii, Acinetobacter baumannii, Acinetobacter calcoaceticus. All the isolates possess arsenic accumulation potential and high molecular weight plasmid (>10 kb). PCR amplification indicated the presence of arsenic-resistance genes (arsB and aoxB) either in the genome or plasmid or in both in the isolated bacteria (except in Acinetobacter venetianus). Exposure to arsenic affected bacterial growth and induced alteration in cytoplasmic membrane integrity.

Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages

Mycobacterium fortuitum causes ‘mycobacteriosis’ in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

Calmodulin–CaMKII mediated alteration of oxidative stress: interplay of the cAMP/PKA–ERK 1/2-NF-κB–NO axis on arsenic-induced head kidney macrophage apoptosis

Using the head kidney macrophages (HKM) from catfish (Clarias batrachus) we earlier demonstrated the role of calcium (Ca2+) and its dependent neutral protease calpain in arsenic-induced apoptosis. Here, we report the role of the CaM–CaMKII axis as an initiator of the process. With the help of specific assay kits and inhibitors we document the pro-apoptotic role of CaM and CaMKII in arsenic-induced HKM apoptosis. CaM-induced CaMKII activity influenced superoxide ion production in exposed cells with a consequent increase in intracellular cAMP levels. Using H-89, the specific inhibitor for PKA, we show for the first time the pro-apoptotic role of the cAMP/PKA pathway in arsenic-induced HKM apoptosis. We report the cAMP/PKA pathway to be critical for initiating downstream activation of MAPKs, namely ERK 1/2. The superoxide ions generated due to arsenic-stress also induce NF-κB activation in HKM. Inducible NOS activity and consequent NO production were evident in the exposed HKM and our study implicates the involvement of ERK 1/2 and NF-κB in the process. Arsenic exposure alters mitochondrial membrane potential, releases cytochrome C and activates caspase-9 leading to caspase-3 mediated apoptosis of HKM. Our findings, thus, provide insight into the underlying mechanism of arsenic toxicity and indicate that HKM could serve as an important in vitro model for immunotoxicity assays.

Identification of novel signature genes attesting arsenic-induced immune alterations in adult zebrafish (Danio rerio)

Arsenic poisoning is a serious global issue. Apart from causing developmental and systemic toxicity, arsenic has recently been reported for its ability to hinder immune responses. The present study is designed to identify the global expression profile associated with arsenic-induced immune alterations at the organismic level. Adult zebrafish (Danio rerio) were exposed to 20, 40 and 80ppb of arsenic trioxide for 30days, sacrificed and global gene expression profile studied. Microarray data suggested 65 immune related genes were commonly affected in the three treatment regimens. The expression profile of key immune related genes (tlr1, nitr1f, nitr1c, crfb8, socs7, socs3b, abcb3/1, mch1uja, ifnγ1-2, cxcl12b and crlf1a) was validated by qPCR. Pathway analysis suggested the major involvement of JAK-STAT circuit in the process. The expression of these marker genes was also studied in arsenic exposed and bacteria (Aeromonas hydrophila) challenged zebrafish. Increase in bacterial colony forming units (CFU) coupled with gross histopathology of kidney in arsenic exposed-bacteria challenged fish suggested profound immuno-compromised condition. We propose that chronic arsenic exposure leads to hyperactivation of the immune system as a consequence when exposed to further stress (microbial) it induces immuno-suppression with pathological implications. The study provides a molecular snap shot for predicting arsenic immuno-toxicity.

Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections

Abstract The present study describes the immunotoxic effect of chronic fluoride exposure on adult zebrafish (Danio rerio). Zebrafish were exposed to fluoride (71.12 mg/L; 1/10 LC50) for 30 d and the expression of selected genes studied. We observed significant elevation in the detoxification pathway gene cyp1a suggesting chronic exposure to non‐lethal concentration of fluoride is indeed toxic to fish. Fluoride mediated pro‐oxidative stress is implicated with the downregulation in superoxide dismutase 1 and 2 (sod1/2) genes. Fluoride affected DNA repair machinery by abrogating the expression of the DNA repair gene rad51 and growth arrest and DNA damage inducible beta a gene gadd45ba. The upregulated expression of casp3a coupled with altered Bcl‐2 associated X protein/B‐cell lymphoma 2 ratio (baxa/bcl2a) clearly suggested chronic fluoride exposure induced the apoptotic cascade in zebrafish. Fluoride‐exposed zebrafish when challenged with non‐lethal dose of fish pathogen A. hydrophila revealed gross histopathology in spleen, bacterial persistence and significant mortality. We report that fluoride interferes with system‐level output of pro‐inflammatory cytokines tumour necrosis factor‐&agr;, interleukin‐1&bgr; and interferon‐&ggr;, as a consequence, bacteria replicate efficiently causing significant fish mortality. We conclude, chronic fluoride exposure impairs the redox balance, affects DNA repair machinery with pro‐apoptotic implications and suppresses pro‐inflammatory cytokines expression abrogating host immunity to bacterial infections. HighlightsChronic fluoride exposure induces histopathological changes in spleen of zebrafish.Fluoride modulates cyp1a, sod1/2, rad51, gadd45ba, baxa/bcl2a and casp3a expression.Fluoride diminishes pro‐inflammatory cytokine response in zebrafish.Fluoride leads to immunosuppression rendering zebrafish susceptible to bacterial infections.

Aeromonas hydrophila-induced alterations in cytosolic calcium activate pro-apoptotic cPKC-MEK1/2-TNFα axis in infected headkidney macrophages of Clarias gariepinus

Abstract Alterations in intracellular‐calcium (Ca2+)i homeostasis is critical to Aeromonas hydrophila‐induced headkidney macrophages (HKM) apoptosis of Clarias gariepinus, though the implications are poorly understood. Here, we describe the role of intermediate molecules of Ca2+−signaling pathway that are involved in HKM apoptosis. We observed phosphoinositide‐3‐kinase/phospholipase C is critical for (Ca2+)i release in infected HKM. Heightened protein kinase‐C (PKC) activity and phosphorylation of MEK1/2‐ERK1/2 was noted which declined in presence of 2‐APB, Go6976 and PD98059, inhibitors to IP3‐receptor, conventional PKC isoforms (cPKC) and MEK1/2 respectively implicating Ca2+/cPKC/MEK‐ERK1/2 axis imperative in A. hydrophila‐induced HKM apoptosis. Significant tumor necrosis factor‐&agr; (TNF&agr;) production and its subsequent reduction in presence of MEK‐ERK1/2 inhibitor U0126 suggested TNF&agr; production downstream to cPKC‐mediated signaling via MEK1/2‐ERK1/2 pathway. RNAi and inhibitor studies established the role of TNF&agr; in inducing caspase‐8‐mediated apoptosis of infected HKM. We conclude, alterations in A. hydrophila‐induced (Ca2+)i alterations activate cPKC‐MEK1/2‐ERK1/2‐TNF&agr; signaling cascade triggering HKM apoptosis. HighlightsTyrosine phosphorylation is indispensable for phagocytosis of A. hydrophila by HKM.PI‐3K/PLC signaling is important for altered cytosolic Ca2+ levels in infected HKM.cPKC‐MEK‐ERK1/2 axis plays pro‐apoptotic role in A. hydrophila‐pathogenesis.A. hydrophila induces TNF&agr;‐mediated caspase‐8 dependent apoptosis of HKM.