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

Shelly, Asha; Banerjee, Chaitali; Saurav, Gunjan Kumar; Ray, Atish; Rana, Vipin Singh; Rajagopal, Raman; Mazumder, Shibnath

doi:10.1016/j.dci.2017.07.015

Cited by 12 publications

(16 citation statements)

References 45 publications

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“…Fish were fed boiled chicken liver ad libitum and acclimatized to laboratory conditions for 15 d before use for experimental purposes. The water quality and fish health in tanks was regularly monitored 57 .…”

Section: Methodsmentioning

confidence: 99%

“…HKM (1 × 10 6 ) pre-treated with or without inhibitors or transfected with sc-siRNA or specific siRNAs were incubated with 2 µM Fluo-3/AM (Invitrogen) for 1 h in the dark. HKM were washed to remove excess dye, infected with M. smegmatis and the changes in fluorescence intensity measured at indicated time points on a microplate fluorimeter (Spectramax M2, Molecular Devices) at excitation A 485 and emission A 520 57 . Tp was used as positive control in the assay.…”

Section: Methodsmentioning

confidence: 99%

“…The cell-free culture supernatant was collected 24 h p.i. and TNF-α levels measured with fish specific TNF-α ELISA kit using reagents supplied with the kit as described earlier 57 .…”

Section: Methodsmentioning

confidence: 99%

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TLR-2 mediated cytosolic-Ca2+ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-α production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages

Hussain

Datta

Singh

et al. 2019

Sci Rep

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The implications of TLR-2 mediated alterations in cytosolic-Ca 2+ ((Ca 2+ ) c ) levels in M . smegmatis infections is not well known. Using headkidney macrophages (HKM) from Clarias gariepinus , we observed TLR-2 signalling is required in the phagocytosis of M . smegmatis . M . smegmatis induced caspase-dependent HKM apoptosis in MOI, time and growth-phase dependent manner. RNAi and inhibitor studies demonstrated critical role of TLR-2 in eliciting (Ca 2+ ) c -surge and c-Src-PI3K-PLC axis playing an intermediary role in the process. The (Ca 2+ ) c -surge triggered downstream ER-stress and superoxide (O 2 − ) generation. The cross-talk between ER-stress and O 2 − amplified TNF-α production, which led to HKM apoptosis and bacterial clearance. Release of nitric oxide (NO) was also observed and silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase activity. Pre-treatment with diphenyleneidonium chloride inhibited NO production implicating O 2 −– NO axis imperative in M . smegmatis- induced HKM apoptosis. NO positively impacted CHOP expression and TNF-α production in infected HKM. We conclude that, TLR-2 induced (Ca 2+ ) c -surge and ensuing cross-talk between ER-stress and O 2 − potentiates HKM pathology by amplifying pro-inflammatory TNF-α production. Moreover, the pro-oxidant environment triggers NO release which prolonged ER-stress and TNF-α production, culminating in HKM apoptosis and bacterial clearance. Together, our study suggests HKM an alternate model to study macrophage-mycobacteria interactions.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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TLR-2 mediated cytosolic-Ca2+ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-α production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages

Hussain

Datta

Singh

et al. 2019

Sci Rep

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“…TNF-a is well known to induce the activation of caspase-8 under various conditions of stress (49,50). We had previously observed that M. fortuitum triggers caspase-8-mediated apoptosis of HKM (36).…”

Section: Tnf-a Affects Hkm Apoptosis and Clearance Of M Fortuitummentioning

confidence: 96%

mtROS Induced via TLR-2-SOCE Signaling Plays Proapoptotic and Bactericidal Role in Mycobacterium fortuitum-Infected Head Kidney Macrophages of Clarias gariepinus

2021

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The mechanisms underlying Mycobacterium fortuitum-induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (Clarias gariepinus), we report that Ca2+ surge across mitochondrial-Ca2+ uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2–endoplasmic reticulum (ER) stress–store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca2+/mtROS cascade in M. fortuitum-infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of CHOP, STIM1, and Orai1, which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to M. fortuitum infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that M. fortuitum-induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance.

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“…collected and stored at −20°C for determination of interferon-ɣ (IFNɣ; # MBS011958), interleukin-1β (IL-1 β, # MBS700230)(El-Houseiny et al, 2021) and tumour necrosis factorα (TNFα, # MBS704369)(Shelly et al, 2017) through ELISA following the protocol of the manufacturer (MyBioSource). The kit used to measure TNFα was a competitive ELISA kit where the plate was pre-coated with a specific antibody for TNFα.…”

mentioning

confidence: 99%

Mitigating Aeromonas hydrophila infection in Nile tilapia through dietary Basella alba and Withania somnifera supplementation: A bioassay‐guided fractionation approach

Mukherjee

Ghosal

Marik

et al. 2022

Aquaculture Research

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The present study aimed to identify the antibacterial bioactive compounds in Withania roots and Basella leaves against Aeromonas infection in Nile tilapia. Withania ethanol extract (EW, ZoI 10.72 ± 1.028 mm) and Basella methanol extract (MB, ZoI 10.83 ± 1.058 mm) showed the highest antibacterial activity in disc diffusion assay.In in vivo experiment, Nile tilapia (25.61 ± 0.31 g) were fed basal, EW and MB (both at 0.7 g/kg feed) diets for 15 days either before being intraperitoneally injected with Aeromonas and basal diet feeding for 7 days (prophylactic) or after bacterial injection and basal diet feeding for 7 days (therapeutic). Fish fed EW and MB fortified diets showed significantly (p < 0.05) lower mortality, alkaline phosphatase, glutamic oxaloacetic transaminase, glutamic pyruvate transaminase activities, HSP70, Caspase3, IFN-ɣ, IL-1 β, TNFα level, while significantly (p < 0.05) higher weight gain, protein efficiency ratio, respiratory burst, lysozyme, phagocytotic activity, plasma total protein, total immunoglobulin content, RBC, WBC count, haemoglobin, haematocrit, mean corpuscular haemoglobin concentration, mean corpuscular volume, lymphocytes, neutrophils, monocytes percentages and superoxide dismutase, catalase, glutathione peroxidase activities compared to basal diet-fed fish. EW showed significantly (p < 0.05) better antibacterial efficacy compared to MB. Prophylactic treatment showed better results compared to therapeutic treatment. Bioassay-guided chromatography and GC-MS analysis revealed the presence of hexadecanoic acid, phytol, cis, cis, cis-7,10,13-hexadecatrienal in EW and α-tocopherol, 1-(+)-ascorbic acid 2,6-dihexadecanoate, 1-heptatriacotanol in MB, which was reported to have antibacterial, antioxidative and immunostimulatory potential. Present results indicate prospective use of EW as a prophylactic immunostimulant and effective therapeutic remedy against Aeromonas in Nile tilapia.

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

Cited by 12 publications

References 45 publications

TLR-2 mediated cytosolic-Ca2+ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-α production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages

TLR-2 mediated cytosolic-Ca2+ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-α production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages

mtROS Induced via TLR-2-SOCE Signaling Plays Proapoptotic and Bactericidal Role in Mycobacterium fortuitum-Infected Head Kidney Macrophages of Clarias gariepinus

Mitigating Aeromonas hydrophila infection in Nile tilapia through dietary Basella alba and Withania somnifera supplementation: A bioassay‐guided fractionation approach

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