Browsing by Author "Cobo, Eduardo R."
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Item Open Access Bovine milk-derived extracellular vesicles reduce oxidative stress and ferroptosis induced by Klebsiella pneumoniae in bovine mammary epithelial cells(2025-02-14) Liang, Bingchun; Xiong, Yindi; Cobo, Eduardo R.; Kastelic, John; Tong, Xiaofang; Han, Bo; Gao, JianAbstract Background Ferroptosis is characterized by increased production of reactive oxygen species (ROS) and membrane lipid peroxidation that can exacerbate inflammatory damage. Extracellular vesicles (EVs) isolated from bovine milk have many biological functions, including antioxidant properties. However, the role of EVs on Klebsiella pneumoniae-induced ferroptosis and oxidative stress in bovine mammary epithelial cells (bMECs) and murine mammary tissue is unclear. In this study, EVs were isolated from bovine colostrum, mature milk and clinical mastitis milk (defined as C-EVs, M-EVs and CM-EVs, respectively) and assessed by transmission electron microscopy, Western blot and transcriptome sequencing. Effects of EVs on K. pneumoniae-induced ferroptosis and oxidative stress in bMECs were evaluated with immunofluorescence and Western blot. Results In bMECs, infection with K. pneumoniae induced oxidative stress, decreasing protein expression of Nrf2, Keap1 and HO-1 plus SOD activity, and increasing ROS concentrations. However, protein expression of GPX4, ACSL4 and S100A4 in bMECs, all factors that regulate ferroptosis, was downregulated by K. pneumoniae. Furthermore, this bacterium compromised tight junctions in murine mammary tissue, with low expression of ZO-1 and Occludin, whereas protein expression of Nrf2 and GPX4 was also decreased in mammary tissue. Adding C-EVs, M-EVs or CM-EVs reduced oxidative stress and ferroptosis in K. pneumoniae-infected bMECs in vitro and murine mammary tissues in vivo. Conclusion In conclusion, all 3 sources of milk-derived EVs alleviated oxidative stress and ferroptosis in K. pneumoniae-infected bMECs and mammary tissues.Item Open Access Cathelicidin contributes to prompt protective inflammation against Toxoplasma gondii(2019-07-10) Tan, Yi Lin; Cobo, Eduardo R.; Kastelic, John P.; Gilleard, John Stuart; Finney, Constance A. M.Toxoplasma gondii is an intracellular parasite infecting all warm-blooded animals, including humans. Although infected immunocompetent individuals are usually asymptomatic, in immunocompromised individuals, T. gondii can affect the central nervous system and may cause congenital toxoplasmosis and death. Cathelicidins are short cationic peptides secreted by leukocytes and epithelial cells with antimicrobial and immunomodulatory activities. It has been proposed that cathelicidin might be a key defense against intracellular pathogens. For instance, human cathelicidin (LL-37), either endogenous or synthetic exogenous, reduced survival of intracellular Mycobacterium tuberculosis in macrophages. However, the role of cathelicidin in toxoplasmosis has been barely investigated. The objective was to elucidate the contributions of cathelicidin during acute systemic and long-term infection with T. gondii. In an acute generalized model of toxoplasmosis, C57BL/6 cathelicidin-deficient (Camp-/-) and wild type (Camp+/+) mice were challenged with luciferase-green fluorescence protein tagged T. gondii (high virulence Type 1 RH, 1 x 105, intra-peritoneal). Although all mice had succumbed by day 5 post infection, Camp-/- mice failed to initiate pro-inflammatory responses in vital organs (ileum, colon, liver, spleen and brain; p<0.05) at early infection (1 d). Consequently, more parasite load was detected in those vital organs (p<0.05). In long-term toxoplasmosis, we determined that Camp-/- mice were more susceptible to oral challenge with T. gondii cysts (low-virulence Type 2 ME 49); all Camp-/- infected mice died by day 12 post infection (p<0.05), whereas Camp+/+ infected and PBS treated mice survived throughout the 14 d study. These Camp-/- mice had more severe hepatitis, with evident liver necrosis and increased inflammatory cytokines, Ifn-γ and Tnf-α gene expression, than Camp+/+ mice (p<0.05). In Camp-/- infected mice, there was pronounced inflammation and Ifn-γ gene synthesis in their spleen (p<0.05) and in their ileum, more severe epithelial disruption, with fewer goblet cells. In in vitro studies, human macrophages (THP-1) infected by T. gondii (ME 49) expressed elevated endogenous gene transcriptional cathelicidin (p<0.05) together with induced gene transcriptional expression of IFN-γ and TNF-α (p<0.05). Camp-/- bone marrow derived macrophages (BMMs) challenged with T. gondii (RH or ME 49) secreted more Tnf-α (p<0.05) compared to infected Camp+/+ BMMs. The T. gondii burdens (both RH and ME 49 strains) were similar between Camp+/+ and Camp-/- BMMs. However, when THP-1 cells macrophages were pre-stimulated with recombinant human LL-37 cathelicidin (2 uM), T. gondii burden was reduced (p<0.05). In summary, this study identified the critical role of cathelicidin in initiating host immune responses, promptly during toxoplasmosis. In the absence of cathelicidin, inflammatory responses in vital organs (liver and spleen) were exaggerated at later infection (3 and 14 d) and they were detrimental to the host. Immunomodulatory roles of cathelicidin were manifested in infected macrophages, where the peptide supressed exaggerated inflammatory responses and aided macrophage killing capabilities. Cathelicidin secreted by macrophages during T. gondii infection has a vital role in host-pathogen balance, controlling parasite burden and preventing overwhelming inflammatory responses.Item Open Access Exploration of innate immune response during infectious bovine digital dermatitis and the evaluation of topical therapeutic treatment(2018-09-18) Watts, Kaitlyn; Cobo, Eduardo R.; Barkema, Herman W.; De Buck, Jeroen M.; Jenne, Craig N.Digital dermatitis (DD) is a frequently occurring infectious disease amongst dairy cattle associated with ulcerative and necrotizing lesions. Due to the associated pain and lameness, DD is a recognized animal welfare problem and has economic implications associated with decreased milk production, lower reproduction rates, and premature culling. DD is of polymicrobial etiology, with the main causative agent identified as belonging to the Treponema genus. Current treatments include topical application of antibiotics such as oxytetracycline or foot baths containing caustic chemicals; however clinical cure rates remain highly variable. In this thesis cattle with DD were monitored to explore the skin innate immune response. An exhaustive description of the inflammatory response during disease progression and a novel description of bovine host defence peptides (HDPs) and their contribution to disease resolution are found herein. It was observed that active DD was characterized by necrotic tissue populated with neutrophils and elevated Cxcl-8 and Tlr4 expression. Tracheal antimicrobial peptide (Tap) was vastly increased in active lesions and key for the resolution of DD. An in vitro model utilizing human keratinocytes showed pro-inflammatory cytokines are released in the absence of living treponemes through Tlr2 signaling and that secretory treponeme products induced cathelicidins. The ability to manipulate inflammatory reactions via treatment with vitamin D3 in DD was compared to the commonly-used oxytetracycline. A cohort of cattle with M2 were topically treated with vitamin D3 against powdered oxytetracycline for 5 days. Although vitamin D3 did elevated Tap expression, lesions and inflammatory markers remained unchanged. In contrast, oxytetracycline reduced neutrophil chemoattractant Cxcl-8 while Tlr2 remained elevated. Histologic assessment evidenced scab formation. Taken together, this thesis established the skin innate response and role of host defence peptides (Tap) during DD and supported oxytetracycline as a treatment, providing lesion resolution and aiding in bacterial elimination.Item Open Access Immuno-Modulating Properties of Tulathromycin in Porcine Reproductive and Respiratory Syndrome Virus-Infected Macrophages In Vitro.(2018-08-10) Desmonts de Lamache, Dimitri; Buret, Andre G.; Morck, Douglas W.; Yates, Robin M.; Cobo, Eduardo R.With a total cost of productivity losses estimated at $600 million annually in the U.S alone, porcine reproductive and respiratory syndrome (PRRS) is a major concern in the swine industry. PRRS etiological agent, the porcine reproductive and respiratory syndrome virus (PRRSV) is a small positive-strand RNA virus that primarily grows in alveolar macrophages. Due to its high antigenic variability, and poorly understood immunopathogenesis, there is currently no treatment to control PRRSV infection. Commercially available vaccines are inefficient and cannot meet practical needs encouraging more researchers to explore different approaches to treat PRRSV infections. The common occurrence of PRRSV infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics for the treatment of the disease it causes. Tulathromycin (TUL), a macrolide antibiotic previously studied in our laboratory has been shown to exhibit potent anti-inflammatory and immunomodulatory actions in cattle and pigs. The aim of this study was to identify and characterize anti-viral and immunomodulating properties of TUL in PRRSV-infected porcine macrophages. Our findings indicate that blood monocyte-derived macrophages are readily infected by PRRSV and can be used as a cellular model to study PRRSV pathogenesis. TUL was found to not change viral titers and viral receptors (CD163 and CD169) expression suggesting that the drug does not possess direct antiviral effects against PRRSV. In addition, we showed that TUL acts synergistically with PRRSV to induce apoptosis but prevents virus-induced early necrosis. TUL was also found to attenuate PRRSV-induced macrophage pro-inflammatory signaling (CXCL-8 and mitochondrial ROS production) and prevent phagocytosis inhibition. Together, these data demonstrate that tulathromycin downregulates PRRSV-induced inflammatory response in macrophages which may in turn reduce virus-related tissue injury. More importantly, this study sheds the light on the potential clinical benefits of an antibiotic in the context of a virus-induced inflammation.Item Open Access Immunomodulatory Role of Cathelicidin at Colonic Epithelium(2020-03-23) Holani, Ravi; Cobo, Eduardo R.; Proud, David; Barkema, Herman W.; Vogel, Hans J.Cathelicidin, a small, cationic and amphiphilic host defense peptide, is expressed by hematopoietic and non-hematopoietic cells. Initially considered an antimicrobial, cathelicidin are more than simply natural antibiotics. Due to their ability to interact with multiple receptors, cathelicidin can modulate immune responses by a variety of host cells. However, their immunomodulatory effects vary based on their micro-environment, including tissue type (hematopoietic vs. non-hematopoietic), availability of microbes or associated factor(s) and effective concentration of cathelicidin. Since most studies are performed in leukocytes, ability of cathelicidin to regulate immune responses by intestinal epithelium remains largely unknown. The objective was to elucidate immunomodulatory potential of cathelicidin in colonic epithelium, under homeostasis as well as during bacterial colitis. In the first study, we determined how cathelicidin synergized with lipopolysaccharide or Salmonella enterica Typhimurium to enhance production of neutrophil chemokine CXCL8 (human) or murine CXCL1 (functional homologue of CXCL8). Further, we identified a two-signal mechanism triggered by the complex of LPS-LL37, in which nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling was involved in CXCL8 mRNA synthesis, whereas p38 mitogen-activated protein kinase (p38MAPK) was involved in CXCL8 mRNA stabilization. In a Citrobacter rodentium model of colitis, we corroborated increased synthesis of CXCL1 chemokine, effective neutrophil recruitment and reduced bacterial burden in Camp+/+ versus Camp-/- (cathelicidin null) mice. In the second study, under physiological conditions, cathelicidin induced synthesis of Toll-like receptor (TLR) negative regulator, Toll-interacting protein (Tollip). This induced Tollip production inhibited TLR-mediated apoptosis in colonic epithelium, both in vitro by cytokines tumour necrosis factor- α and interferon- γ as well as in vivo in a C. rodentium model of colitis. In conclusion, cathelicidin regulated colonic innate immunity via reduction of unwarranted inflammation by inhibiting TLR responses through Tollip, while promoting a neutrophil response to infection.Item Open Access NK cell killing of extracellular Pseudomonas aeruginosa(2020-06-26) Feehan, David; Mody, Christopher Hugh; Cobo, Eduardo R.; Harrison, Joe J.Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects individuals with the respiratory illness, Cystic Fibrosis and contributes to airway blockage and loss of lung function. NK cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes. The hypothesis of this project is that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co¬-culture of YT cells, a human NK cell line, and GFP P. aeruginosa stained with the membrane viability dye propidium iodide, demonstrated that YT cell killing of P. aeruginosa is contact mediated. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa, as determined by CFU count. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of PAO1 in comparison to wild type controls. This demonstrates that granzymes are required for killing, but exhibit redundancy in their function. In summary, these results suggest that NK cell kill P. aeruginosa through a membrane damaging, contact-dependent process, requiring granzymes.