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

Immunomodulation of human T cell responses with receptor selective enkephalins

The δ-opioid receptor selective [2-d-penicillamine-5-d-penicillamine] enkephalin (DPDPE) and the μ receptor selective Tyr-d-Orn-Phe-As p-NH2 (TOPA) were found respectively, to have marked immunostimulant and immunosuppressant activities in both normal subjects and patients suffering from leprosy and tuberculosis. Antigen specific lymphoproliferation and numbers of rosette forming T cells were significantly (P <0.05) enhanced on in vitro treatment with Met-enkephalin. This was further increased (P <0.001) in the presence of the d selective DPDPE. In contrast, treatment with μ selective TOPA inhibited lymphoproliferation substantially (P <0.01) and rosette formation to a lesser extent

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

Complete Nucleotide Sequence of a Quinolone Resistance Gene (qnrS2) Carrying Plasmid of Aeromonas Hydrophila Isolated from Fish.

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment

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

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

<div><p>The role of calcium (Ca²⁺) and its dependent protease calpain in <i>Aeromonas hydrophila</i>-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKII<i>g</i>) in the process. We observed significant increase in CaM levels in <i>A. hydrophila</i>-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 CaMKII<i>g</i>. Using the CaMKII<i>g</i>-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKII<i>g</i> signalling to be critical for apoptosis of <i>A. hydrophila</i>-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKII<i>g</i> expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in <i>A. hydrophila</i>-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 CaMKII<i>g</i> 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 <i>A. hydrophila</i>. 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 <i>A. hydrophila</i> activates CaM and calpain-2; both pathways converge on CaMKII<i>g</i> which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.</p></div

List of degenerate primers.

<p>List of degenerate primers.</p

PKA is pro-apoptotic in <i>A. hydrophila</i>-infected HKM.

<p>(A) HKM were transfected with CaMKII<i>g</i>-siRNA, PKACA-siRNA or scrambled siRNA then infected with <i>A. hydrophila</i> and PKACA mRNA expression detected by real time PCR 2 h p.i. HKM were pre-treated with H-89 or transfected with PKACA-siRNA or scrambled siRNA then infected with <i>A. hydrophila</i> and checked for (B) Hoechst 33342 positive cells and caspase-3 activity and (C) AV-PI staining 24 h p.i. (D) <i>A. hydrophila</i> viability was checked following either pre-incubation of HKM with H-89, co-incubation with 8-Br-cAMP or transfection with PKACA-siRNA. The intracellular bacterial number was determined by dilution plating on nutrient agar plate. Vertical bars represent mean ± SE (n = 6). <b>*</b>P<0.05, compared to HKM; <b>^γ</b>P<0.05, compared to HKM+Sc; <b>^#</b>P<0.05, compared to HKM+B; <b>^±</b>P<0.05, compared to HKM+Sc+B. HKM, control head kidney macrophage; HKM+Sc, HKM transfected with scrambled siRNA; HKM+B, HKM infected with <i>A. hydrophila</i>; HKM+Sc+B, HKM transfected with scrambled siRNA infected with <i>A. hydrophila</i>; HKM+CaMKII<i>g</i>-siRNA+B, HKM transfected with CaMKII<i>g</i>-siRNA infected with <i>A. hydrophila</i>; HKM+PKACA-siRNA+B, HKM transfected with PKACA-siRNA infected with <i>A. hydrophila</i>; HKM+H-89+B, HKM pre-treated with H-89 infected with <i>A. hydrophila</i>; HKM+8-Br-cAMP+B, HKM exposed to 8-Br-cAMP infected with <i>A. hydrophila</i>.</p

CaM-CaMKII-cAMP/PKA-ERK 1/2 axis leads to caspase-3 mediated apoptosis of <i>A. hydrophila</i>-infected HKM.

<p><i>A. hydrophila</i>-elevated Ca²⁺ leads to downstream calpain-2 activation and CaM expression. The pathways converge at CaMKII<i>g</i> to induce cAMP/PKA mediated activation of ERK 1/2 leading to caspase-3 mediated apoptosis of <i>A. hydrophila</i>-infected HKM.</p