Mitochondrial electron transport chain in macrophage reprogramming: Potential role in antibacterial immune response.

Macrophages restrain microbial infection and reinstate tissue homeostasis. The mitochondria govern macrophage metabolism and serve as pivot in innate immunity, thus acting as immunometabolic regulon. Metabolic pathways produce electron flows that end up in mitochondrial electron transport chain (mtETC), made of super-complexes regulating multitude of molecular and biochemical processes. Cell-intrinsic and extrinsic factors influence mtETC structure and function, impacting several aspects of macrophage immunity. These factors provide the macrophages with alternate fuel sources and metabolites, critical to gain functional competence and overcoming pathogenic stress. Mitochondrial reactive oxygen species (mtROS) and oxidative phosphorylation (OXPHOS) generated through the mtETC are important innate immune attributes, which help macrophages in mounting antibacterial responses. Recent studies have demonstrated the role of mtETC in governing mitochondrial dynamics and macrophage polarization (M1/M2). M1 macrophages are important for containing bacterial pathogens and M2 macrophages promote tissue repair and wound healing. Thus, mitochondrial bioenergetics and metabolism are intimately coupled with innate immunity. In this review, we have addressed mtETC function as innate rheostats that regulate macrophage reprogramming and innate immune responses. Advancement in this field encourages further exploration and provides potential novel macrophage-based therapeutic targets to control unsolicited inflammation.

Role of UPRmt and mitochondrial dynamics in host immunity: it takes two to tango.

The immune system of a host contains a group of heterogeneous cells with the prime aim of restraining pathogenic infection and maintaining homeostasis. Recent reports have proved that the various subtypes of immune cells exploit distinct metabolic programs for their functioning. Mitochondria are central signaling organelles regulating a range of cellular activities including metabolic reprogramming and immune homeostasis which eventually decree the immunological fate of the host under pathogenic stress. Emerging evidence suggests that following bacterial infection, innate immune cells undergo profound metabolic switching to restrain and countervail the bacterial pathogens, promote inflammation and restore tissue homeostasis. On the other hand, bacterial pathogens affect mitochondrial structure and functions to evade host immunity and influence their intracellular survival. Mitochondria employ several mechanisms to overcome bacterial stress of which mitochondrial UPR (UPRmt) and mitochondrial dynamics are critical. This review discusses the latest advances in our understanding of the immune functions of mitochondria against bacterial infection, particularly the mechanisms of mitochondrial UPRmt and mitochondrial dynamics and their involvement in host immunity.

ß-Catenin Elicits Drp1-Mediated Mitochondrial Fission Activating the Pro-Apoptotic Caspase-1/IL-1ß Signalosome in Aeromonas hydrophila-Infected Zebrafish Macrophages.

Canonical Wnt signaling plays a major role in regulating microbial pathogenesis. However, to date, its involvement in A. hydrophila infection is not well known. Using zebrafish (Danio rerio) kidney macrophages (ZKM), we report that A. hydrophila infection upregulates wnt2, wnt3a, fzd5, lrp6, and ß-catenin (ctnnb1) expression, coinciding with the decreased expression of gsk3b and axin. Additionally, increased nuclear ß-catenin protein accumulation was observed in infected ZKM, thereby suggesting the activation of canonical Wnt signaling in A. hydrophila infection. Our studies with the ß-catenin specific inhibitor JW67 demonstrated ß-catenin to be pro-apoptotic, which initiates the apoptosis of A. hydrophila-infected ZKM. ß-catenin induces NADPH oxidase (NOX)-mediated ROS production, which orchestrates sustained mitochondrial ROS (mtROS) generation in the infected ZKM. Elevated mtROS favors the dissipation of the mitochondrial membrane potential (ΔΨm) and downstream Drp1-mediated mitochondrial fission, leading to cytochrome c release. We also report that ß-catenin-induced mitochondrial fission is an upstream regulator of the caspase-1/IL-1ß signalosome, which triggers the caspase-3 mediated apoptosis of the ZKM as well as A. hydrophila clearance. This is the first study suggesting a host-centric role of canonical Wnt signaling pathway in A. hydrophila pathogenesis wherein ß-catenin plays a primal role in activating the mitochondrial fission machinery, which actively promotes ZKM apoptosis and helps in containing the bacteria.

TLR22-Induced Pro-Apoptotic mtROS Abets UPRmt-Mediated Mitochondrial Fission in Aeromonas hydrophila-Infected Headkidney Macrophages of Clarias gariepinus.

Toll-like receptors (TLRs) are epitomized as the first line of defense against pathogens. Amongst TLRs, TLR22 is expressed in non-mammalian aquatic vertebrates, including fish. Using headkidney macrophages (HKM) of Clarias gariepinus, we reported the pro-apoptotic and microbicidal role of TLR22 in Aeromonas hydrophila infection. Mitochondria act as a central scaffold in the innate immune system. However, the precise molecular mechanisms underlying TLR22 signaling and mitochondrial involvement in A. hydrophila-pathogenesis remain unexplored in fish. The aim of the present study was to investigate the nexus between TLR22 and mitochondria in pro-apoptotic immune signaling circuitry in A. hydrophila-infected HKM. We report that TLR22-induced mitochondrial-Ca2+ [Ca2+]mt surge is imperative for mtROS production in A. hydrophila-infected HKM. Mitigating mtROS production enhanced intracellular bacterial replication implicating its anti-microbial role in A. hydrophila-pathogenesis. Enhanced mtROS triggers hif1a expression leading to prolonged chop expression. CHOP prompts mitochondrial unfolded protein response (UPRmt) leading to the enhanced expression of mitochondrial fission marker dnml1, implicating mitochondrial fission in A. hydrophila pathogenesis. Inhibition of mitochondrial fission reduced HKM apoptosis and increased the bacterial burden. Additionally, TLR22-mediated alterations in mitochondrial architecture impair mitochondrial function (ΔΨm loss and cytosolic accumulation of cyt c), which in turn activates caspase-9/caspase-3 axis in A. hydrophila-infected HKM. Based on these findings we conclude that TLR22 prompts mtROS generation, which activates the HIF-1α/CHOP signalosome triggering UPRmt-induced mitochondrial fragmentation culminating in caspase-9/-3-mediated HKM apoptosis and bacterial clearance.

Th1-Th2 and M1-M2 interplay sculpt Aeromonas hydrophila pathogenesis in zebrafish (Danio rerio).

Aeromonas hydrophila is an important aquatic zoonotic pathogen that causes septicemia, necrotizing fasciitis and gastroenteritis in various aquatic and non-aquatic animals. However, the pathogenesis of A. hydrophila is not fully understood. Here, we examined the pathogenicity and histopathology of A. hydrophila in the zebrafish (Danio rerio) model system. We found that the intensity of symptoms and mortality is dose-dependent. Bacterial colonization studies demonstrated that A. hydrophila never cleared out from the fish body but stayed in a state of inactivity till it enters a fresh host. Reinfection studies showed that exposure to A. hydrophila provides immunity against future infection and hence improves fish survival. Gene expression studies revealed the crosstalk between T-helper cell and macrophage responses in fish immune system in response to A. hydrophila and infection memory. Histopathological studies showed that symptoms of tissue damage and inflammation lasted for less duration with less intensity in immunized fish when compared to non-immunized fish. Together, our results suggest that the zebrafish model is a useful system in studying the interplay between A. hydrophila pathogenesis, persistence and immunity.

Aeromonas hydrophila inhibits autophagy triggering cytosolic translocation of mtDNA which activates the pro-apoptotic caspase-1/IL-1ß-nitric oxide axis in headkidney macrophages.

The molecular mechanisms underlying Aeromonas hydrophila-pathogenesis are not well understood. Using head kidney macrophages (HKM) of Clarias gariepinus, we previously reported the role of ER-stress in A. hydrophila-induced pathogenesis. Here, we report that PI3K/PLC-induced cytosolic-Ca2+ imbalance induces the expression of pro-apoptotic ER-stress marker, CHOP in A. hydrophila-infected HKM. CHOP promotes HKM apoptosis by inhibiting AKT activation and enhancing JNK signaling. Elevated mitochondrial ROS (mtROS) was recorded which declined significantly by ameliorating ER-stress and in the presence of ER-Ca2+ release modulators (2-APB and dantrolene) and mitochondrial-Ca2+ uptake inhibitor, Ru360, together suggesting the role of ER-mitochondrial Ca2+ dynamics in mtROS generation. Inhibiting mtROS production reduced HKM death implicating the pro-apoptotic role of mtROS in A. hydrophila-pathogenesis. The expression of autophagic proteins (LC3B, beclin-1, and atg 5) was suppressed in the infected HKM. Our results with autophagy-inducer rapamycin demonstrated that impaired autophagy favored the cytosolic accumulation of mitochondrial DNA (mtDNA) and the process depended on mtROS levels. Enhanced caspase-1 activity and IL-1ß production was detected and transfection studies coupled with pharmacological inhibitors implicated mtROS/mtDNA axis to be crucial for activating the caspase-1/IL-1ß cascade in infected HKM. RNAi studies further suggested the involvement of IL-1ß in generating pro-apoptotic NO in A. hydrophila-infected HKM. Our study suggests a novel role of ER-mitochondria cross-talk in regulating A. hydrophila pathogenesis. Based on our observations, we conclude that A. hydrophila induces ER-stress and inhibits mitophagy resulting in mitochondrial dysfunction which leads to mtROS production and translocation of mtDNA into cytosol triggering the activation of caspase-1/IL-1ß-mediated NO production, culminating in HKM apoptosis.

TLR22-mediated activation of TNF-α-caspase-1/IL-1ß inflammatory axis leads to apoptosis of Aeromonas hydrophila-infected macrophages.

Toll-like receptors (TLRs) represent first line of host defence against microbes. Amongst different TLRs, TLR22 is exclusively expressed in non-mammalian vertebrates, including fish. The precise role of TLR22 in fish-immunity remains abstruse. Herein, we used headkidney macrophages (HKM) from Clarias gariepinus and deciphered its role in fish-immunity. Highest tlr22 expression was observed in the immunocompetent organ - headkidney; nonetheless expression in other tissues suggests its possible involvement in non-immune sites also. Aeromonas hydrophila infection up-regulates tlr22 expression in HKM. Our RNAi based study suggested TLR22 restricts intracellular survival of A. hydrophila. Inhibitor and RNAi studies further implicated TLR22 induces pro-inflammatory cytokines TNF-α and IL-1ß. We observed heightened caspase-1 activity and our results suggest the role of TLR22 in activating TNF-α/caspase-1/IL-1ß cascade leading to caspase-3 mediated apoptosis of A. hydrophila-infected HKM. We conclude, TLR22 plays critical role in immune-surveillance and triggers pro-inflammatory cytokines leading to caspase mediated HKM apoptosis and pathogen clearance.

M. fortuitum-induced CNS-pathology: Deciphering the role of canonical Wnt signaling, blood brain barrier components and cytokines.

Molecular underpinning of mycobacteria-induced CNS-pathology is not well understood. In the present study, zebrafish were infected with Mycobacterium fortuitum and the prognosis of CNS-pathogenesis studied. We observed M. fortuitum triggers extensive brain-pathology. Evans blue extravasation demonstrated compromised blood-brain barrier (BBB) integrity. Further, decreased expression in tight-junction (TJ) and adherens junction complex (AJC) genes were noted in infected brain. Wnt-signaling has emerged as a major player in host-mycobacterial immunity but its involvement/role in brain-infection is not well studied. Sustained expression of wnt2, wnt3a, fzd5, lrp5/6 and ß-catenin, with concordant decline in degradation complex components axin, gsk3ß and ß-catenin regulator capn2a were observed. The surge in ifng1 and tnfa expression preceding il10 and il4 suggested cytokine-interplay critical in M. fortuitum-induced brain-pathology. Therefore, we suggest adult zebrafish as a viable model for studying CNS-pathology and using the same, conclude that M. fortuitum infection is associated with repressed TJ-AJC gene expression and compromised BBB permeability. Our results implicate Wnt/ß-catenin pathway in M. fortuitum-induced CNS-pathology wherein Th1-type signals facilitate bacterial clearance and Th2-type signals prevent the disease sequel.

TLRs in Mycobacterial Pathogenesis: Black and White or Shades of Gray.

Toll-like receptors (TLRs) play critical role in the innate recognition of pathogens besides orchestrating innate and adaptive immune responses. These receptors exhibit exquisite specificity for different pathogens or their products and, through a complex network of signalling, generate appropriate immune responses. TLRs induce both pro- and anti-inflammatory signals depending on interactions with the adapter molecules thereby impacting the outcome of infection. Hence, TLR signalling ought to be stringently regulated to avoid harmful effects on the host. Mycobacteria express antigens which are sensed by TLRs leading to activation of various signalling molecules important for initiating the death of infected cells and containment of pathogens. Conversely, it also utilizes TLRs for immune evasion and persistence. Due to the enormous diversity in the repertoire of virulence traits expressed by mycobacteria, genetic variations in TLRs often impair the host's ability to respond to mycobacterial-stress, affecting health and disease manifestations. Thus, understanding TLR signalling is of great importance for insights into host-mycobacterial interactions and designing effective measures for controlling the spread and persistence of the bacterium.

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

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.

The coordinated outcome of STIM1-Orai1 and superoxide signalling is crucial for headkidney macrophage apoptosis and clearance of Mycobacterium fortuitum.

The mechanisms underlying M. fortuitum-induced pathogenesis remains elusive. Using headkidney macrophages (HKM) from Clarias gariepinus, we report that TLR-2-mediated internalization of M. fortuitum is imperative to the induction of pathogenic effects. Inhibiting TLR-2 signalling alleviated HKM apoptosis, thereby favouring bacterial survival. Additionally, TLR-2-mediated cytosolic calcium (Ca2+)c elevation was instrumental for eliciting ER-stress in infected HKM. ER-stress triggered the activation of membrane-proximal calcium entry channels comprising stromal interaction molecule 1 (STIM1) and calcium-release activated calcium channel 1 (Orai1). RNAi studies suggested STIM1-Orai1 signalling initiate calpain-mediated cleavage of nitric oxide synthase interacting protein, prompting the release of pro-apoptotic nitric oxide. Inhibiting STIM1-Orai1 signalling attenuated superoxide production (O2•-) and vice versa. We conclude, TLR-2-induced ER-stress triggers STIM1/Orai1 expression and that the reciprocal association between STIM1-Orai1 signalling and oxidative stress is critical for sustaining (Ca2+)c level, thereby prolonging ER-stress and maintenance of pro-oxidant rich environment to induce HKM apoptosis and bacterial clearance.

Arsenic induced alteration in Mrp-1 like activity leads to zebrafish hepatocyte apoptosis: The cellular GSH connection.

Multidrug-resistance protein-1 facilitates the efflux of arsenic conjugated with reduced glutathione nonetheless; the relation between Mrp-1 ATPase activity and cellular GSH levels is contentious. To study this, Mrp-1-ATPase activity was measured in 5 µM arsenic trioxide exposed zebrafish hepatocytes (ZFH) and correlated with intracellular GSH levels. Alongside, mrp-1 gene expression as well as Mrp-1 protein level was also monitored. Diverse mode of Mrp-1 inhibition was reflected from differential level of Km and Vmax of Mrp-1 at different time points. 3 h post-arsenic treatment demonstrated non-competitive inhibition. At 6 h, there was significant increase in Km and ZFH death, suggesting reduced binding affinity of Mrp-1 for ATP. Increased caspase-9-cytochromeC-ATP levels (putative apoptosome), reinforced ZFH apoptosis. The increase in Vmax coupled with reduced substrate affinity of Mrp-1 suggests malfunctioning in arsenic- tolerance mechanisms. We posit the triggering glutathione level regulate arsenic tolerance in ZFH. Irreversible impairment of ATP binding to Mrp-1 culminates in arsenic-induced ZFH apoptosis.

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.

The implications of TLR-2 mediated alterations in cytosolic-Ca2+((Ca2+)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 (Ca2+)c-surge and c-Src-PI3K-PLC axis playing an intermediary role in the process. The (Ca2+)c-surge triggered downstream ER-stress and superoxide (O2-) generation. The cross-talk between ER-stress and O2- 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 O2--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 (Ca2+)c-surge and ensuing cross-talk between ER-stress and O2- 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.

Tryptophan-kynurenine pathway attenuates ß-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection.

Recent studies have documented the diverse role of host immunity in infection by the protozoan parasite, Toxoplasma gondii. However, the contribution of the ß-catenin pathway in this process has not been explored. Here, we show that AKT-mediated phosphorylated ß-catenin supports T. gondii multiplication which is arrested in the deficiency of its phosphorylation domain at S552 position. The ß-catenin-TCF4 protein complex binds to the promoter region of IRF3 gene and initiates its transcription, which was also abrogated in ß-catenin knockout cells. TBK-independent phosphorylation of STING(S366) and its adaptor molecule TICAM2 by phospho-AKT(T308S473) augmented downstream IRF3-dependent IDO1 transcription, which was also dependent on ß-catenin. But, proteasomal degradation of IDO1 by its tyrosine phosphorylation (at Y115 and Y253) favoured parasite replication. In absence of IDO1, tryptophan was catabolized into melatonin, which supressed cellular reactive oxygen species (ROS) and boosted parasite growth. Conversely, when tyrosine phosphorylation was abolished by phosphosite mutations, IDO1 escaped its ubiquitin-mediated proteasomal degradation system (UPS) and the stable IDO1 prevented parasite replication by kynurenine synthesis. We propose that T. gondii selectively utilizes tryptophan to produce the antioxidant, melatonin, thus prolonging the survival of infected cells through functional AKT and ß-catenin activity for better parasite replication. Stable IDO1 in the presence of IFN-γ catabolized tryptophan into kynurenine, promoting cell death by suppressing phospho-AKT and phospho-ß-catenin levels, and circumvented parasite replication. Treatment of infected cells with kynurenine or its analogue, teriflunomide suppressed kinase activity of AKT, and phosphorylation of ß-catenin triggering caspase-3 dependent apoptosis of infected cells to inhibit parasite growth. Our results demonstrate that ß-catenin regulate phosphorylated STING-TICAM2-IRF3-IDO1 signalosome for a cell-intrinsic pro-parasitic role. We propose that the downstream IRF3-IDO1-reliant tryptophan catabolites and their analogues can act as effective immunotherapeutic molecules to control T. gondii replication by impairing the AKT and ß-catenin axis.

Mycobacterium fortuitum-induced ER-Mitochondrial calcium dynamics promotes calpain/caspase-12/caspase-9 mediated apoptosis in fish macrophages.

Mycobacterium fortuitum is a natural fish pathogen. It induces apoptosis in headkidney macrophages (HKM) of catfish, Clarias sp though the mechanism remains largely unknown. We observed M. fortuitum triggers calcium (Ca2+) insult in the sub-cellular compartments which elicits pro-apototic ER-stress factor CHOP. Alleviating ER-stress inhibited CHOP and attenuated HKM apoptosis implicating ER-stress in the pathogenesis of M. fortuitum. ER-stress promoted calpain activation and silencing the protease inhibited caspase-12 activation. The study documents the primal role of calpain/caspase-12 axis on caspase-9 activation in M. fortuitum-pathogenesis. Mobilization of Ca2+ from ER to mitochondria led to increased mitochondrial Ca2+ (Ca2+)m load,, mitochondrial permeability transition (MPT) pore opening, altered mitochondrial membrane potential (ΔΨm) and cytochrome c release eventually activating the caspase-9/-3 cascade. Ultra-structural studies revealed close apposition of ER and mitochondria and pre-treatment with (Ca2+)m-uniporter (MUP) blocker ruthenium red, reduced Ca2+ overload suggesting (Ca2+)m fluxes are MUP-driven and the ER-mitochondria tethering orchestrates the process. This is the first report implicating role of sub-cellular Ca2+ in the pathogenesis of M. fortuitum. We summarize, the dynamics of Ca2+ in sub-cellular compartments incites ER-stress and mitochondrial dysfunction, leading to activation of pro-apoptotic calpain/caspase-12/caspase-9 axis in M. fortuitum-infected HKM.

Aeromonas hydrophila utilizes TLR4 topology for synchronous activation of MyD88 and TRIF to orchestrate anti-inflammatory responses in zebrafish.

Toll-like receptor 4 (TLR4) plays a critical role in host immunity against Gram-negative bacteria. It transduces signals through two distinct TIR-domain-containing adaptors, MyD88 and TRIF, which function at the plasma membrane and endosomes, respectively. Using zebrafish Aeromonas hydrophila infection model, we demonstrate that synchronization of MyD88 and TRIF dependent pathways is critical for determining the fate of infection. Zebrafish were infected with A. hydrophila, and bacterial recovery studies suggested its effective persistence inside the host. Histopathological assessment elucidates that A. hydrophila did not provoke inflammatory responses in the spleen. Immunofluorescence revealed the presence of TLR4-bound A. hydrophila on the plasma membrane at 3 h post-infection (p.i.), and inside endosomes 1 day p.i. Quantitative PCR studies suggest that TLR4 activates the downstream pathway of MyD88-IRAK4 axis at early stages followed by a shift to TRIF-TRAF6 axis at late stages of infection coupled with fold increase in NFκB. Our results implicated the involvement of p110δ isoform of PI(3)Kinase in this transition. Coupled to this, we noted that the TLR4-TRIF-NFκB axis prompted burgeoned secretion of anti-inflammatory cytokines. We observed that A. hydrophila inhibits endosome maturation and escapes to cytoplasm. Significant downregulation of cytosolic-NLR receptors further suggested that A. hydrophila represses pro-inflammatory responses in cytosol aiding its persistence. Our findings suggest a novel role of 'TLR4 topology' in A. hydrophila-induced pathogenesis. We propose that A. hydrophila manipulates translocation of TLR4 and migrates to endosome, where it triggers TRIF-dependent anti-inflammatory responses, interferes with endosomal maturation and escapes to cytosol. Inside the cytosol, A. hydrophila avoids detection by suppressing NLRs, facilitating its survival and ensuing pathogenesis.

Chronic fluoride exposure exacerbates headkidney pathology and causes immune commotion in Clarias gariepinus.

The current study was aimed to understand the effects of chronic fluoride exposure on fish immune system. African sharp tooth catfish (Clarias gariepinus) were exposed to 73.45mg/L of fluoride corresponding to 1/10 96h LC50 for 30 d and the effects on general fish health and several immune parameters were studied. Chronic fluoride exposure led to significant alteration in serum biochemical parameters including alkaline phosphatase, alanine transaminase, aspartate transaminase, triglycerides, cholesterol and blood urea nitrogen levels revealing the detrimental effect of fluoride on general fish health. Upregulation in cytochrome P450 1A expression, both at mRNA and protein level suggested that fluoride activates the detoxification machinery in headkidney (HK) of C. gariepinus. Histopathological analysis of HK from exposed fish further revealed fluoride-induced hypertrophy, increase in melano-macrophage centers (MMCs) and the development of cell-depleted regions. Fluoride reduced headkidney somatic index (HKSI) and the phagocytic potential of headkidney macrophages (HKM). It induced caspase-3-dependent headkidney leukocyte (HKL) apoptosis, elevated superoxide generation and production of pro-inflammatory cytokine TNF-α besides suppressed T-cell proliferation in the exposed fish. We surmise the elevation in superoxide levels coupled with increased TNF-α production to be plausible causes of fluoride-induced HKL apoptosis. It is concluded that chronic fluoride exposure induces structure-function alterations in HK, the primary lymphoid organ in fish leading to impairment in immune responses.

Chronic endosulfan exposure impairs immune response rendering Clarias gariepinus susceptible to microbial infection.

Endosulfan, a persistent organochlorine insecticide affects several off-target organisms including fish though the underlying mechanisms remain obscure. In the present study, we monitored the effect of chronic endosulfan exposure on headkidney (HK), an important immune organ in fish and on fish immune system thereof. Clarias gariepinus were exposed to a non-lethal concentration of endosulfan 2.884ppb (1/10th LC50) for 30 d which resulted in suppressed phagocytosis and bactericidal potential of headkidney macrophages (HKM). The same non-lethal concentration of endosulfan also interfered with T-cell proliferation and serum antibody titer in fish. Endosulfan-exposed fish were challenged with non-lethal dose of fish pathogenic bacteria Aeromonas hydrophila and the 'exposure-challenge' study revealed endosulfan-exposed C. gariepinus severely immunocompromised and prone to bacterial infections. Depuration for 30 d suggested that except for phagocytosis and serum agglutination titer other endosulfan-induced immune aberrations could not be restored significantly. Nonetheless, compared to exposed-challenged fish the depurated fish showed significant improvement in viability on challenge with A. hydrophila. Collectively, these findings suggest chronic endosulfan exposure has prolonged effect on fish making them prone to microbial infections.

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

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-α (TNFα) production and its subsequent reduction in presence of MEK-ERK1/2 inhibitor U0126 suggested TNFα production downstream to cPKC-mediated signaling via MEK1/2-ERK1/2 pathway. RNAi and inhibitor studies established the role of TNFα 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-TNFα signaling cascade triggering HKM apoptosis.

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

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-α, interleukin-1ß and interferon-γ, 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.