Koumiss promotes Mycobacterium bovis infection by disturbing intestinal flora and inhibiting endoplasmic reticulum stress.

Mycobacterium bovis is the causative agent of bovine tuberculosis and also responsible for serious threat to public health. Koumiss is a fermented mare's milk product, used as traditional drink. Here, we explored the effect of koumiss on gut microbiota and the host immune response against M bovis infection. Therefore, mice were treated with koumiss and fresh mare milk for 14 days before M bovis infection and continue for 5 weeks after infection. The results showed a clear change in the intestinal flora of mice treated with koumiss, and the lungs of mice treated with koumiss showed severe edema, inflammatory infiltration, and pulmonary nodules in M bovis-infected mice. Notably, we found that the content of short-chain fatty acids was significantly lower in the koumiss-treated group compared with the control group. However, the expression of endoplasmic reticulum stress and apoptosis-related proteins in the lungs of koumiss-treated mice were significantly decreased. Collectively, these findings suggest that koumiss treatment disturb the intestinal flora of, which is associated with disease severity and the possible mechanism that induces lungs pathology. Our current findings can be exploited further to establish the "gut-lung" axis which might be a novel strategy for the control of tuberculosis.

Mitochondrial Transcription Factor A Regulates Mycobacterium bovis-Induced IFN-ß Production by Modulating Mitochondrial DNA Replication in Macrophages.

BACKGROUND: Mycobacterium bovis persistently survives in macrophages by developing multiple strategies to evade host immune responses, and the early induction of interferon-ß (IFN-ß) is one of these critical strategies. The mitochondrial transcription factor A (TFAM) plays a vital role in mitochondrial DNA (mtDNA) metabolism and has been suggested to influence IFN-ß production in response to viral infection. However, its role in the production of IFN-ß by M. bovis has not been elucidated. METHODS: In the current study, we investigated the role of TFAM in the production of IFN-ß in M. bovis-infected macrophages. RESULTS: We found that knockdown of TFAM expression significantly reduced M. bovis-induced IFN-ß production, mtDNA copy numbers and cytosolic mtDNA were increased in murine macrophages with M. bovis infection, cytosolic mtDNA contributed to IFN-ß production, and TFAM was required for the increase in mtDNA copy numbers induced by M. bovis. We also observed that TFAM affected the intracellular survival of M. bovis. CONCLUSIONS: Our results suggest that TFAM plays an essential role in M. bovis-induced IFN-ß production by regulating mtDNA copy numbers. This might be a new strategy adopted by M. bovis for its intracellular survival.

Inhibition of type I interferon signaling abrogates early Mycobacterium bovis infection.

BACKGROUND: Mycobacterium bovis (M. bovis) is the principal causative agent of bovine tuberculosis; however, it may also cause serious infection in human being. Type I IFN is a key factor in reducing viral multiplication and modulating host immune response against viral infection. However, the regulatory pathways of Type I IFN signaling during M. bovis infection are not yet fully explored. Here, we investigate the role of Type I IFN signaling in the pathogenesis of M. bovis infection in mice. METHODS: C57BL/6 mice were treated with IFNAR1-blocking antibody or Isotype control 24 h before M. bovis infection. After 21 and 84 days of infection, mice were sacrificed and the role of Type I IFN signaling in the pathogenesis of M. bovis was investigated. ELISA and qRT-PCR were performed to detect the expression of Type I IFNs and related genes. Lung lesions induced by M. bovis were assessed by histopathological examination. Viable bacterial count was determined by CFU assay. RESULTS: We observed an abundant expression of Type I IFNs in the serum and lung tissues of M. bovis infected mice. In vivo blockade of Type I IFN signaling reduced the recruitment of neutrophils to the lung tissue, mediated the activation of macrophages leading to an increased pro-inflammatory profile and regulated the inflammatory cytokine production. However, no impact was observed on T cell activation and recruitment in the early acute phase of infection. Additionally, blocking of type I IFN signaling reduced bacterial burden in the infected mice as compared to untreated infected mice. CONCLUSIONS: Altogether, our results reveal that Type I IFN mediates a balance between M. bovis-mediated inflammatory reaction and host defense mechanism. Thus, modulating Type I IFN signaling could be exploited as a therapeutic strategy against a large repertoire of inflammatory disorders including tuberculosis.

PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis-Infected Macrophages.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis in cattle population across the world. Human beings are at equal risk of developing tuberculosis beside a wide range of M. bovis infections in animal species. Autophagic sequestration and degradation of intracellular pathogens is a major innate immune defense mechanism adopted by host cells for the control of intracellular infections. It has been reported previously that the catalytic subunit of protein phosphatase 2A (PP2Ac) is crucial for regulating AMP-activated protein kinase (AMPK)-mediated autophagic signaling pathways, yet its role in tuberculosis is still unclear. Here, we demonstrated that M. bovis infection increased PP2Ac expression in murine macrophages, while nilotinib a tyrosine kinase inhibitor (TKI) significantly suppressed PP2Ac expression. In addition, we observed that TKI-induced AMPK activation was dependent on PP2Ac regulation, indicating the contributory role of PP2Ac towards autophagy induction. Furthermore, we found that the activation of AMPK signaling is vital for the regulating autophagy during M. bovis infection. Finally, the transient inhibition of PP2Ac expression enhanced the inhibitory effect of TKI-nilotinib on intracellular survival and multiplication of M. bovis in macrophages by regulating the host's immune responses. Based on these observations, we suggest that PP2Ac should be exploited as a promising molecular target to intervene in host-pathogen interactions for the development of new therapeutic strategies towards the control of M. bovis infections in humans and animals.

Combinatory FK506 and Minocycline Treatment Alleviates Prion-Induced Neurodegenerative Events via Caspase-Mediated MAPK-NRF2 Pathway.

Transcription factors play a significant role during the symptomatic onset and progression of prion diseases. We previously showed the immunomodulatory and nuclear factor of activated T cells' (NFAT) suppressive effects of an immunosuppressant, FK506, in the symptomatic stage and an antibiotic, minocycline, in the pre-symptomatic stage of prion infection in hamsters. Here we used for the first time, a combinatory FK506+minocycline treatment to test its transcriptional modulating effects in the symptomatic stage of prion infection. Our results indicate that prolonged treatment with FK506+minocycline was effective in alleviating astrogliosis and neuronal death triggered by misfolded prions. Specifically, the combinatory therapy with FK506+minocycline lowered the expression of the astrocytes activation marker GFAP and of the microglial activation marker IBA-1, subsequently reducing the level of pro-inflammatory cytokines interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), and increasing the levels of anti-inflammatory cytokines IL-10 and IL-27. We further found that FK506+minocycline treatment inhibited mitogen-activated protein kinase (MAPK) p38 phosphorylation, NF-kB nuclear translocation, caspase expression, and enhanced phosphorylated cAMP response element-binding protein (pCREB) and phosphorylated Bcl2-associated death promoter (pBAD) levels to reduce cognitive impairment and apoptosis. Interestingly, FK506+minocycline reduced mitochondrial fragmentation and promoted nuclear factor⁻erythroid2-related factor-2 (NRF2)-heme oxygenase 1 (HO-1) pathway to enhance survival. Taken together, our results show that a therapeutic cocktail of FK506+minocycline is an attractive candidate for prolonged use in prion diseases and we encourage its further clinical development as a possible treatment for this disease.

Comparative Study of the Molecular Basis of Pathogenicity of M. bovis Strains in a Mouse Model.

It is widely accepted that different strains of Mycobacterium tuberculosis have variable degrees of pathogenicity and induce different immune responses in infected hosts. Similarly, different strains of Mycobacterium bovis have been identified but there is a lack of information regarding the degree of pathogenicity of these strains and their ability to provoke host immune responses. Therefore, in the current study, we used a mouse model to evaluate various factors involved in the severity of disease progression and the induction of immune responses by two strains of M. bovis isolated from cattle. Mice were infected with both strains of M. bovis at different colony-forming unit (CFU) via inhalation. Gross and histological findings revealed more severe lesions in the lung and spleen of mice infected with M. bovis N strain than those infected with M. bovis C68004 strain. In addition, high levels of interferon-γ (IFN-γ), interleukin-17 (IL-17), and IL-22 production were observed in the serum samples of mice infected with M. bovis N strain. Comparative genomic analysis showed the existence of 750 single nucleotide polymorphisms and 145 small insertions/deletions between the two strains. After matching with the Virulence Factors Database, mutations were found in 29 genes, which relate to 17 virulence factors. Moreover, we found an increased number of virulent factors in M. bovis N strain as compared to M. bovis C68004 strain. Taken together, our data reveal that variation in the level of pathogenicity is due to the mutation in the virulence factors of M. bovis N strain. Therefore, a better understanding of the mechanisms of mutation in the virulence factors will ultimately contribute to the development of new strategies for the control of M. bovis infection.

IFN-ß: A Contentious Player in Host-Pathogen Interaction in Tuberculosis.

Tuberculosis (TB) is a major health threat to the human population worldwide. The etiology of the disease is Mycobacterium tuberculosis (Mtb), a highly successful intracellular pathogen. It has the ability to manipulate the host immune response and to make the intracellular environment suitable for its survival. Many studies have addressed the interactions between the bacteria and the host immune cells as involving many immune mediators and other cellular players. Interferon-ß (IFN-ß) signaling is crucial for inducing the host innate immune response and it is an important determinant in the fate of mycobacterial infection. The role of IFN-ß in protection against viral infections is well established and has been studied for decades, but its role in mycobacterial infections remains much more complicated and debatable. The involvement of IFN-ß in immune evasion mechanisms adopted by Mtb has been an important area of investigation in recent years. These advances have widened our understanding of the pro-bacterial role of IFN-ß in host-pathogen interactions. This pro-bacterial activity of IFN-ß appears to be correlated with its anti-inflammatory characteristics, primarily by antagonizing the production and function of interleukin 1ß (IL-1ß) and interleukin 18 (IL-18) through increased interleukin 10 (IL-10) production and by inhibiting the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome. Furthermore, it also fails to provoke a proper T helper 1 (Th1) response and reduces the expression of major histocompatibility complex II (MHC-II) and interferon-γ receptors (IFNGRs). Here we will review some studies to provide a paradigm for the induction, regulation, and role of IFN-ß in mycobacterial infection. Indeed, recent studies suggest that IFN-ß plays a role in Mtb survival in host cells and its downregulation may be a useful therapeutic strategy to control Mtb infection.

An appraisal of traditional knowledge of plant poisoning of livestock and its validation through acute toxicity assay in rats.

Background: Kashmir Himalaya hosts the most diverse and rich flora in the world, which serves as grazing land for millions of small ruminants in the area. While most plant species are beneficial, some can be poisonous, causing economic losses and animal health issues for livestock. Consequently, this study is the first comprehensive report on the traditional phyto-toxicological knowledge in District Muzaffarabad and the assessment of its authenticity through experimental studies in rats. Methods: The data regarding traditional knowledge was gathered from 70 key respondents through semi-structured interviews, which was quantitatively analyzed and authenticated through plant extract testing on Wistar female rats and comparison with published resources. Results: A total of 46 poisonous plant species belonging to 23 families and 38 genera were reported to be poisonous in the study area. Results revealed that leaves were the most toxic plant parts (24 species, 52.1%), followed by the whole plant (18 species, 39.1%), stem (17 species, 36.9%), and seeds (10 species, 21.7%). At the organ level, liver as most susceptible affected by 13 species (28.2%), followed by the gastrointestinal tract (15 species, 32.6%), nervous system (13 species, 8.2%), dermis (8 species, 17.3%), renal (7 species, 15.2%), respiratory (4 species, 8.7%), cardiovascular system (3 species, 6.5%), and reproductive system (2 species, 4.3%). The poisonous plant species with high Relative frequency citation (RFC) and fidelity level (FL) were Nerium oleander (RFC, 0.6; FL, 100), Lantana camara (RFC, 0.6; FL, 100), and Ricinus communis (RFC, 0.6; FL, 100). Experimental assessment of acute toxicity assay in rats revealed that Nerium oleander was the most toxic plant with LD50 of (4,000 mg/kg), trailed by Ricinus communis (4,200 mg/kg), L. camara (4,500 mg/kg), and Datura stramonium (4,700 mg/kg); however, other plants showed moderate to mild toxicity. The major clinical observations were anorexia, piloerection, dyspnea, salivation, tachypnea, constipation, diarrhea, tremor, itchiness, and dullness. Conclusion: This study showed that numerous poisonous plants pose a significant risk to the livestock industry within Himalayan territory, leading to substantial economic losses. Consequently, it is of utmost importance to conduct further comprehensive studies on the phytotoxicity of plants.

Phytotoxicological study of selected poisonous plants from Azad Jammu & Kashmir.

Poisonous plants cause tremendous economic losses to the livestock industry. These economic losses are deterioration in their health, decreased productivity, deformed offspring, and reduced longevity. The current study is the first comprehensive report on poisonous plants of Azad Jammu and Kashmir which systematically documents the phytotoxicological effect and mode of action in livestock. The information was gathered from 271 informants including 167 men and 104 women through semi-structured interviews and literature search through available databases. The data collected through interviews was analyzed with quantitative tools viz. the factor informant consensus and fidelity level. A total of 38 species of flowering plants belonging to 23 families and 38 genera were reported. Family Asteraceae (5 spp) was the most dominant, followed by Solanaceae (4 spp), Fabaceae (4 spp), Euphorbiaceae (4 spp) and Convolvulaceae (3 spp). Among all the species collected, herbs were the dominant life form (22 spp, 57.89%), trailed by shrubs (11 spp, 28.95%), and trees (5 spp, 13.16%). Whole plant toxicity was reported to be the highest (15 spp, 39.47%), followed by leaf toxicity (12 spp, 31.58%), seed toxicity (4 spp, 7.89%), fruit toxicity (3 spp, 10.53%), latex toxicity (2 spp, 5.26%), flowers toxicity (1 spp, 2.63%), and berries toxicity (1 spp, 2.63%). The most toxic route of administration was found oral (39 spp, 40.63%), followed by intraperitoneal (24 spp, 25%), and intravenous (21 spp, 21.88%). The most commonly affected organ was found liver (20.41%), followed by gastrointestinal tract (20.341%), CNS (16.33%), skin (14.29%), kidneys (12.24%), lungs (4.04%), reproductive organs (2.04%), spleen (1.75%), blood (1.75%), heart (1.75%), urinary tract (1.75%), and pancreas (1.75%). The maximum Fic value was found for dermatological disorders (0.91), followed by the endocrine system (0.90), gastrointestinal (0.82), neurology (0.77), nephrology (0.67), cardiovascular (0.67), urinary (0.67), respiratory (0.60), sexual (0.60) disorders. Senecio vulgaris, and Ageratum conyzoides were the most important plants with fidelity level (0.95) and (0.87). Nerium oleander, Lantana camara, Leucaena leucocephala, and Ricinus communis were the important poisonous plant with maximum fidelity level (100%). Ricinus communis with reported lowest LD50 (<20 mg/kg) was the top-ranked poisonous plant followed by Lantana camara and Justicia adhatoda (25-50 mg/kg), Nerium Oleander (157.37 mg/kg), and Datura innoxia (400 mg/kg). We found that knowledge about poisonous plants is less prevailing in the rural areas of Azad Kashmir compared to the knowledge about medicinal plants and poisonous nature of reported plants is due to production of toxic substances and presence of essential oils.

Effects of Flaxseed and Multi-Carbohydrase Enzymes on the Cecal Microbiota and Liver Inflammation of Laying Hens.

BACKGROUND: The use of wheat and flaxseed to produce omega-3 (ω-3) enriched poultry meat and eggs is very popular in the world. However, wheat and flaxseed contain some anti-nutritional factors (ANFs), and enzymes are usually used to alleviate the deleterious influence of ANFs. METHOD: A 2 × 3 two factors design was used in the experiment. A total of 540 twenty-week-old Nongda-3 laying hens were randomly allocated to six dietary treatments, two diets (corn/flaxseed and wheat/flaxseed), and three enzymes (enzyme-a contains neutral protease 10,000, xylanase 35,000, ß-mannanase 1500, ß-glucanase 2000, cellulose 500, amylase 100, and pectinase 10,000 (U g-1); enzyme-b contains alkaline protease 40,000 and neutral protease 10,000 (U g-1); enzyme-c contains alkaline protease 40,000, neutral protease 10,000, and cellulase 4000 (U g-1). RESULTS: There was an interaction between dietary treatment and supplemental enzymes for liver weight and liver inflammatory cytokines of broilers. A significant increase was observed in the fat weight of birds fed a corn diet as compared with a wheat diet. A corn diet and wheat diet with the addition of enzyme-a (p < 0.001) showed the highest level of liver fat followed by enzyme-c (p < 0.01) and enzyme-b. Moreover, a high level of secretory IL-1ß, IL-6, and IL-10 and comparatively higher inflammatory changes in the liver tissue were found in birds fed a corn diet as compared with a wheat diet, and enzyme-b showed more beneficial effects as compared with enzyme-a and -c. The gut microbial composition of hens fed a corn diet was significantly different than that of birds fed a wheat diet. Bacteroides were significantly (p < 0.05) abundant in the corn-fed birds as compared with wheat-fed birds. However, Firmicutes were less abundant in the wheat-fed birds than the corn-fed birds (16.99 vs. 31.80%, respectively). The microbial community at the genus level differed significantly in the dietary groups and we observed that Bacteroides are the predominant cecal microbiota. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of co-factors, carbohydrates, vitamins, protein, and energy were expressed at slightly higher levels in the microbiota of the wheat-fed birds, whereas, metabolic pathways for nucleotides, lipids, and glycine were expressed at higher levels in the wheat-fed birds. Furthermore, expression of the growth and cellular processes pathway and endocrine system pathway levels were predicted to be higher for the wheat-fed group as compared with the corn-fed group. CONCLUSIONS: In conclusion, our findings suggest that inflammatory changes in laying birds were mediated by a corn diet with flaxseed and enzymes instead of a wheat diet. Additionally, in the wheat-fed group, enzyme-b and -c showed more encouraging results as compared to enzyme-a.

Caspase-1 inhibits IFN-ß production via cleavage of cGAS during M. bovis infection.

Mycobacterium bovis (M. bovis) infection triggers cytokine production via pattern recognition receptors. These cytokines include type I interferons (IFNs) and interleukin-1ß (IL-1ß). Excessive type I IFN levels impair host resistance to M. bovis infection. Therefore, strict control of type I IFN production is helpful to reduce pathological damage and bacterial burden. Here, we found that a deficiency in caspase-1, which is the critical component of the inflammasome responsible for IL-1ß production, resulted in increased IFN-ß production upon M. bovis infection. Subsequent experiments demonstrated that caspase-1 activation reduced cyclic GMP-AMP synthase (cGAS) expression, thereby inhibiting downstream TANK-binding kinase 1 (TBK1)- interferon regulatory factor 3 (IRF3) signaling and ultimately reducing IFN production. A deficiency in caspase-1 activation enhanced the bacterial burden during M. bovis infection in vitro and in vivo and aggravated pathological lesion formation. Thus, caspase-1 activation reduced IFN-ß production upon M. bovis infection by dampening cGAS-TBK1-IRF3 signaling, suggesting that the inflammasome protects hosts by negatively regulating harmful cytokines.

In vitro Impact of Yeast Expressed Hybrid Peptide CATH-2TP5 as a Prophylactic Measure Toward Sepsis and Inflammation.

CATH-2TP5 is a linear cationic hybrid peptide, consequent from naturally occurring antimicrobial peptide (AMPs) Cathelicidin-2 (CATH-2) and Immunomodulatory peptide Thymopentin (TP5) having dynamic and potent anti-inflammatory activities without hemolytic effect. The biocompatible mechanism of CATH-2TP5 is favored to explore new methodologies in the direction of biomedical applications. In this retrospectively study, an antiendotoxin and anti-inflammatory hybrid peptide CATH-2TP5 was emulated into pPICZα-A and successfully expressed in Pichia pastoris (P. pastoris). The recombinant CATH-2TP5 was purified through the Ni-affinity column and reversed-phase HPLC. The purified CATH-2TP5 peptide exhibited robust anti-endotoxin activity and significantly (p < 0.05) neutralized the effect of lipopolysaccharide (LPS). Furthermore, the down-regulated effect of CATH-2TP was more pronounced (p < 0.05) on LPS-induced cytotoxic effects, nitric oxide secretion and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in murine RAW264.7 macrophages. As associated to control and parental peptide the number of apoptotic cells was also contracted with the treatment of CATH-2TP5. Thus, we concluded that CATH-2TP5 peptide may be used in various biomedical applications as a therapeutic drug.

Matrix metalloproteinases: Expression, regulation and role in the immunopathology of tuberculosis.

Mycobacterium tuberculosis (Mtb) leads to approximately 1.5 million human deaths every year. In pulmonary tuberculosis (TB), Mtb must drive host tissue destruction to cause pulmonary cavitation and dissemination in the tissues. Matrix metalloproteinases (MMPs) are endopeptidases capable of degrading all components of pulmonary extracellular matrix (ECM). It is well established that Mtb infection leads to upregulation of MMPs and also causes disturbance in the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs), thus altering the extracellular matrix deposition. In TB, secretion of MMPs is mainly regulated by NF-κB, p38 and MAPK signalling pathways. In addition, recent studies have demonstrated the immunomodulatory roles of MMPs in Mtb pathogenesis. Researchers have proposed a new regimen of improved TB treatment by inhibition of MMP activity to hinder matrix destruction and to minimize the TB-associated morbidity and mortality. The proposed regimen involves adjunctive use of MMP inhibitors such as doxycycline, marimastat and other related drugs along with front-line anti-TB drugs to reduce granuloma formation and bacterial load. These findings implicate the possible addition of economical and well-tolerated MMP inhibitors to current multidrug regimens as an attractive mean to increase the drug potency. Here, we will summarize the recent advancements regarding expression of MMPs in TB, their immunomodulatory role, as well as their potential as therapeutic targets to control the deadly disease.

Endoplasmic Reticulum Stress Induces Macrophages to Produce IL-1ß During Mycobacterium bovis Infection via a Positive Feedback Loop Between Mitochondrial Damage and Inflammasome Activation.

Mycobacterium bovis, the causative agent of tuberculosis in cattle and humans, infects host macrophages and induces endoplasmic reticulum stress (ERS), mitochondrial damage, and interleukin (IL)-1ß production. The relationship between these phenotypes is yet to be elucidated. In this study, we investigated the role of ERS in mitochondrial damage and IL-1ß production in macrophages during infection with a virulent M. bovis strain. We found that ERS activates the inflammasome via NOD-like receptor family, pyrin domain-containing 3 (NLRP3)-caspase-8 and that IFN-inducible protein absent in melanoma 2 (AIM2) triggered mitochondrial damage. ERS increased reactive oxygen species (ROS), which promoted translocation of the inflammasome to the mitochondria. NLRP3, but not AIM2, was involved in the ERS-induced cleavage of caspase-8 and Bid, leading to mitochondrial damage, which was required for the production of mature IL-1ß. Our data suggest that ERS induces macrophages to produce mature IL-1ß during infection with virulent M. bovis through a positive feedback loop between mitochondrial damage and inflammasome activation. To the best of our knowledge, this is the first evidence of the involvement of ERS and mitochondrial damage in inflammasome activation during M. bovis infection.

Kallikrein 12 Regulates Innate Resistance of Murine Macrophages against Mycobacterium bovis Infection by Modulating Autophagy and Apoptosis.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1ß, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-ß expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.

Nilotinib: A Tyrosine Kinase Inhibitor Mediates Resistance to Intracellular Mycobacterium Via Regulating Autophagy.

Nilotinib, a tyrosine kinase inhibitor, has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. Mycobacterium bovis (M. bovis) and Mycobacterium avium subspecies paratuberculosis (MAP) are the etiological agents of bovine tuberculosis and Johne's disease, respectively. Although M. bovis and MAP cause distinct tissue tropism, both of them infect, reside, and replicate in mononuclear phagocytic cells of the infected host. Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. Here we demonstrated that nilotinib significantly inhibited the intracellular survival and growth of M. bovis and MAP in macrophages by modulating host immune responses. We showed that nilotinib induced autophagic degradation of intracellular mycobacterium occurred via the inhibition of PI3k/Akt/mTOR axis mediated by abelson (c-ABL) tyrosine kinase. In addition, we observed that nilotinib promoted ubiquitin accumulation around M. bovis through activation of E3 ubiquitin ligase parkin. From in-vivo experiments, we found that nilotinib effectively controlled M. bovis growth and survival through enhanced parkin activity in infected mice. Altogether, our data showed that nilotinib regulates protective innate immune responses against intracellular mycobacterium, both in-vitro and in-vivo, and can be exploited as a novel therapeutic remedy for the control of M. bovis and MAP infections.

Regulation of MicroRNAs-Mediated Autophagic Flux: A New Regulatory Avenue for Neurodegenerative Diseases With Focus on Prion Diseases.

Prion diseases are fatal neurological disorders affecting various mammalian species including humans. Lack of proper diagnostic tools and non-availability of therapeutic remedies are hindering the control strategies for prion diseases. MicroRNAs (miRNAs) are abundant endogenous short non-coding essential RNA molecules that negatively regulate the target genes after transcription. Several biological processes depend on miRNAs, and altered profiles of these miRNAs are potential biomarkers for various neurodegenerative diseases, including prion diseases. Autophagic flux degrades the misfolded prion proteins to reduce chronic endoplasmic reticulum stress and enhance cell survival. Recent evidence suggests that specific miRNAs target and regulate the autophagic mechanism, which is critical for alleviating cellular stress. miRNAs-mediated regulation of these specific proteins involved in the autophagy represents a new target with highly significant therapeutic prospects. Here, we will briefly describe the biology of miRNAs, the use of miRNAs as potential biomarkers with their credibility, the regulatory mechanism of miRNAs in major neurodegenerative diseases such as Alzheimer's, Parkinson's, and prion diseases, degradation pathways for aggregated prion proteins, the role of autophagy in prion diseases. Finally, we will discuss the miRNAs-modulated autophagic flux in neurodegenerative diseases and employ them as potential therapeutic intervention strategy in prion diseases.

p62-Keap1-NRF2-ARE Pathway: A Contentious Player for Selective Targeting of Autophagy, Oxidative Stress and Mitochondrial Dysfunction in Prion Diseases.

Prion diseases are a group of fatal and debilitating neurodegenerative diseases affecting humans and animal species. The conversion of a non-pathogenic normal cellular protein (PrPc) into an abnormal infectious, protease-resistant, pathogenic form prion protein scrapie (PrPSc), is considered the etiology of these diseases. PrPSc accumulates in the affected individual's brain in the form of extracellular plaques. The molecular pathways leading to neuronal cell death in prion diseases are still unclear. The free radical damage, oxidative stress and mitochondrial dysfunction play a key role in the pathogenesis of the various neurodegenerative disorders including prion diseases. The brain is very sensitive to changes in the redox status. It has been demonstrated that PrPc behaves as an antioxidant, while the neurotoxic prion peptide PrPSc increases hydrogen peroxide toxicity in the neuronal cultures leading to mitochondrial dysfunction and cell death. The nuclear factor erythroid 2-related factor 2 (NRF2) is an oxidative responsive pathway and a guardian of lifespan, which protect the cells from free radical stress-mediated cell death. The reduced glutathione, a major small molecule antioxidant present in all mammalian cells, and produced by several downstream target genes of NRF2, counterbalances the mitochondrial reactive oxygen species (ROS) production. In recent years, it has emerged that the ubiquitin-binding protein, p62-mediated induction of autophagy, is crucial for NRF2 activation and elimination of mitochondrial dysfunction and oxidative stress. The current review article, focuses on the role of NRF2 pathway in prion diseases to mitigate the disease progression.

A study on prevalence and molecular characterization of trypanosomal species infecting equines in Lahore region, Pakistan.

Trypanosomiasis is an important protozoal disease with a diverse range of susceptible host including human. In the current study, molecular characterization of prevalent species was done through a pan-trypanosome polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). A total of three hundred (n = 300) equines including horses, donkeys and mules (100 each) were randomly selected and the equine blood samples were subjected to screening for trypanosomes through microhaematocrit centrifuge technique (MHCT), conventional PCR, semi-nested PCR and RFLP. Overall prevalence of trypanosomal species was 8% (24/300) as revealed by MHCT and species wise prevalence in horses, donkeys and mules was 4.33% (13/300), 1.33% (4/300) and 2.33% (7/300), respectively. Conventional and semi-nested PCR depicted an overall prevalence of 21% (63/300) and species wise prevalence in horses, donkeys and mules was 12% (36/300), 3.67% (11/300) and 5.33% (16/300), respectively. RFLP analysis of the semi-nested products, using Msp1 and Eco571 enzymes, negated the presence of T. congolense, T. brucei, T. vivax, T. theileri, and T. vivax in the positive samples and revealed that the animals might be suffering from T. evansi infection as the enzymes used were not able to detect this species. This hypothesis was further confirmed by using T. evansi specific primers which depicted all of the 63 samples were positive for T. evansi. It is inferred that T. evansi is the major trypanosome species prevalent in equines. Furthermore, PCR is more sensitive as compared to microscopic examination and the pan-trypanosome PCR-RFLP assay is suitable for carrying out laboratory diagnosis of field samples and epidemiological studies. Further studies on the possibilities of use of other restriction enzymes may help to improve the species specificity of the assay.

miRNAs in Tuberculosis: New Avenues for Diagnosis and Host-Directed Therapy.

Tuberculosis (TB) is one of the most fatal infectious diseases and a leading cause of mortality, with 95% of these deaths occurring in developing countries. The causative agent, Mycobacterium tuberculosis (Mtb), has a well-established ability to circumvent the host's immune system for its intracellular survival. microRNAs (miRNAs) are small, non-coding RNAs having an important function at the post-transcriptional level and are involved in shaping immunity by regulating the repertoire of genes expressed in immune cells. It has been established in recent studies that the innate immune response against TB is significantly regulated by miRNAs. Moreover, differential expression of miRNA in Mtb infection can reflect the disease progression and may help distinguish between active and latent TB infection (LTBI). These findings encouraged the application of miRNAs as potential biomarkers. Similarly, active participation of miRNAs in modulation of autophagy and apoptosis responses against Mtb opens an exciting avenue for the exploitation of miRNAs as host directed therapy (HDT) against TB. Nanoparticles mediated delivery of miRNAs to treat various diseases has been reported and this technology has a great potential to be used in TB. In reality, this exploitation of miRNAs as biomarkers and in HDT is still in its infancy stage, and more studies using animal models mimicking human TB are advocated to assess the role of miRNAs as biomarkers and therapeutic targets. In this review, we attempt to summarize the recent advancements in the role of miRNAs in TB as immune modulator, miRNAs' capability to distinguish between active and latent TB and, finally, usage of miRNAs as therapeutic targets against TB.