BS7101: Advanced Technique In Bioscience - Inflammation Is A Complex Physiological Response - University of East London - Nursing Assessment Answer

INTRODUCTION: Inflammation is a complex physiological response.

First, what is Inflammation? So, the Inflammation is the natural defence mechanism of our body. Immune system of human body recognizes the injured cells or tissues, pathogens and foreign particles presence in the body and started self- protection to remove harmful particles or healing the injured cells. Apart from this positive side, in some situation, there are some side effects also occurred like rubor (redness), calor (heat), tumor (swelling) and dolor (pain).

There are 2 types of inflammation: 1 is acute (primary immune response)

2ndis chronic inflammation

In the Inflammation process, Prostaglandin plays an important role to produce cyclooxygenase and peroxidase enzymes. Cyclooxygenase also known COX enzymes, which has isoforms of COX-1 and COX-2. COX-1 is most influential source of prostanoids and has a functions like gastric epithelial cytoprotection and homeostasis, where COX-2 generate by inflammatory pathogens, growth and hormones factors, is crucial source of prostanoid formation in the process of inflammation and in proliferative diseases like cancer. Prostanoid production is switched by COX enzymes, that covert arachidonic acid into unstable intermediate prostaglandin H2 (PGH

2), which is further converted in cell-specific pathways to numbers of different prostaglandins, prostacyclin and thromboxane. COX-1 and COX-2 are isoforms, their structures are similar, that catalyse identical reactions, but different in expression and their biological roles.

The COX enzymes are target elements for all non-steroidal anti-inflammatory drugs (NSAID). These drugs inhibit the activity of COX and prevent the production of prostanoid. Prostaglandins are an important modulators of immune system. COX-2 play an important role in the induction and resolution of acute inflammation, also expressed in sterile inflammation.

COX-2 is the key element in many normal physiological functions and pathological conditions. It would be the great achievement to have the ability to regulate the production of COX-2 at specific time and in specific cell types. to get this ability, researchers used RNA interference (RNAi) technique in mice model. This achievement should become a important tool to analyse the cell type-specific roles for COX-2. In this technique, short hairpin RNA (shRNA) embedded within a miRNA backbone, this miRNA is processed with the shRNA consisting of a sequence of 21-29 nicleotides and reverse complement of 21-29 nucleotides region. This short RNA folds back by itself and form a hairpin structure, which made a cleave into double-stranded RNAs by an endogenous nuclease Dicer. At the end, RNAi regulates sequence-specific mRNA destruction.

Macrophages have phenotype that expressed their protective and potential role in chronic inflammation and tissue injury. This kind of heterogeneity arises as differentiation of monocytes into macrophages and exposed to hematopoietic cell-derived stimuli and specific tissue, so these macrophages are classified as alternatively or classically activated. This classical activation (M1) occurs in type 1 cytokine or recognition of molecular patterns of pathogens. Thus, M1 is an important for production of immune response against pathogens. Macrophage phenotype revealed cells from nonresolving model M1, where resolution-phase of macrophages alternatively activated with high level of COX-2 expression.

In the recent past, role of human SIRT1 in inflammation and regulator of oxidative stress has been demonstrated. SIRT1 regulates the gene expression of proinflammation with COX-2 as an anti-inflammatory regulatory protein. With this information, scientists evaluate the possibility of bacterial survival limitations in macrophages using a combination of COX-2 inhibitor and an antibiotic, ampicillin via SIRT1, resulted in reduced activity of bacteria.

REMARKS: Given basic information, which helps to understand the process that what will be next step, but there are some questions that unanswered. It will be the positive side, if writer gives more information about shRNA, RNAi and SIRT1.

METHODOLOGY:

(Reversible Suppression of COX-2 Expression IN VIVO by Inducible RNA Interference)

COX-2 shRNA design and cloning:

shRNAs used as a potential gene silencing, to target the COX-2 activity. These predicted shRNAs screened and cross-checked against a sensor exclusion criteria and a transcript database to avoid off-taget gene similarity. Four 22-mer predicted shRNAs (cox2.284, cox2.1082,cox2.2058,cox2.3711). these shRNA sequences and their corresponding sense strand synthesized as 97 mers and cloned into miR30 shRNA backbone. These sequences consist of gene-specific stem and 19 bp loop of the miR30 to create miR30-adapted shRNAs specific for COX-2.

Retrovirus vector preparation and shRNA testing:

The LMP retroviral vector contains XhoI and EcoRI sites in a miR30-shRNA expression cassette, directed by viral 5LTR promoter. The four XhoI/EcoRI COX-2-shRNAs cloned into LMP vector, the retroviral stocks and retroviral control for COX-2-shRNA targeting the firefly luciferase coding sequence, which was generated by VSV-G plasmid transfection in HEK293 cells. To check the ability of that four cox-2-shRNAs to block the induction of COX-2, NIH3T3 cells (mouse embryonic fibroblast cells) and RAW264.7 cells (mouse monocytic leukemia cells) transduced with viral infection. RAW264.7 cells maintained in RPMI 1640 medium containing FBS and penicillin-streptomycin. COX-2 was generated in RAW264.7 cells by adding bacterial LPS. NIH3T3 cells uphold with FBS overnight to generate COX-2 expression. For analysis of prostaglandin E2, culture was harvested, frozen and assayed by ELISA method.

Generation of the TRE-shRNA transgenic mouse:

Mice were generated by using RMCE method (recombinase mediated cassette exchange), cells contained homing cassette downstream of ColA1 gene on mouse chromosome 11. To get site-specific integration into homing cassette, scientists used FLPe recombinase-mediated recombination technique between FRT-sites in ColA1 gene and in the targeting vector. The Cox-2.2058 shRNA in LMP shRNA expression cassette cloned into backbone miR30 of targeting vector at XhoI/EcoRI single site. The Cox2.2058 shRNA containing targeting vector and pCAGs-Flpe were co-electroporated into KH2 ES cells. This combination confirmed the resistance of hygromycin in ESC clones.

Isolation and Culture of mouse Primary bone marrow macrophages and fibroblasts:

Macrophages: Bone marrow macrophages obtained from tibia, pelvis and femur, washed, plated into RPMI medium containing FBS and penicillin/Streptomycin and cultured. Cells treated with LPS for activation of COX2 gene.

Fibroblasts: Fibroblasts tissues collected from ling and skin, treated with liberase and cultured in DMEM/F12 medium to allow fibroblasts to migrate from fragments. COX-2 and luciferase expression generated and analysed as NIH3T3 cells.

COX-2 gene activation in IN VIVO and in Mouse paw:

Control mice injected with saline, after 6 hours of injection, mice were euthanized. Tissues were removed drastically, placed in culture dishes and used for luciferase-dependent bioluminescence imaging. Tissues kept frozen for further examination. Inflammation generated by sub-plantar Zymosan injection into left hind paw, right hind paw was used as control with saline injection. To examine the COX-2 expression, mice were anesthetized by isoflurane inhalation and skin was collected and processed for Western blotting.

Further examination such as Luciferase activity assays, Western blotting and measurement of leukocyte infiltration, different methods were used.

(Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP)

Induction of Peritonitis: Peritonitis generated by intraperitoneal injection of 3,5-cyclic monophosphorothioate 24 hours prior of macrophage isolation. For two-dimensional electrophoresis, cell-free peritoneal obtained by Centricon YM-10 spin column. Albumin was removed by albumin removal column. Electrophoresis in the second dimension, performed after acrylamide gels applied.

Silver staining and Mass spectrometry: One and 2-dimensional gels fixed by incubation in acetic acid and silver stained by use of mass spectrometric compatible protocol. Bands of interest were obtained from gel and got reduction by DTT, alkylation and dehydration in acetonitrile. This dehydrated gel was rehydrated by trypsin. Trypsin digests spotted into target with matrix and peptide masses were determined by MALDI-TOF mass spectrometer. For examination of HMGB1, antibodies and blocking peptides, Western blotting and FACS were used.

For determination of Cytokines and Chemokines, Multiplex cytokine array protocol used. To measured cAMP, enzyme immunoassay used. To obtained the data about proliferated rM or M1 cells, FACS method used.

IN VITRO cell stimulation and Bacterial killing assay:

Staphylococcus aureus (serotype V) bacteria grown in LB and Streptococcus grown in Bacto Todd Hewitt broth. Peritoneal cells were obtained after several steps of incubation, in the presence of penicillin/streptomycin. To measure the ability of macrophage bacterial killing, S aureus added to appropriate wells, centrifuged and incubated in agar plates for 24 hours. For labelling of macrophage, macrophage-specific stain PKH26-PCL or PKH2-PCL injected in peritoneal cavity of mice and confirmed by using FACS.

(Celecoxib Sensitizes Staphylococcus Aureus to Antibiotics in Macrophages by Modulating SIRT1)

S aureus (gram positive bacteria) grown in Brain-heart infusion medium. Mouse macrophage( RAW 264.7) collected from provider. Phagocytosis of S aureus by RAW 264.7 and cells were obtained, grown in gentamycin for elimination of extracellular bacteria. Cells now incubated with or without celecoxib, ampicillin or both in RPMI medium without antibiotic. Assessed the viability of intracellular bacteria after incubation by CFU in LB agar plates.

Western blot method and FACS analysis done to check the activation of TLR2.

Immunoprecipitation and activity of SIRT1 examined by Fluorimetric activity assay kit by treated S aureus with celecoxib, ampicillin, combination of suramin and anti-SIRT1 antibody. The p65 levels of NF-kB measured in nuclear isolates and cytoplasm of S aureus-phagocytosed RAW 264.7 cells treated with or without drugs. SIRT1 activity was checked by Fluor-de-lys SIRT1 kit. Release 0f Cytokines such as MIP-1 alpha, IL-2 etc. analysed by Quantikine immunoassays kits.

REMARKS: In all methods, basic information is missing like which material was used, information about different kits used in experiments, which instruments were used in kits etc. It may be more helpful to understand the exact methodology.

RESULTS:

(Reversible Suppression of COX-2 Expression IN VIVO by Inducible RNAi)

From the improved prediction methods, scientists identified four 22-mer guide strand sequences (COX2.284, COX2.1082, COX2.2058 and COX2.3711), with additional COX2 coding region. Specific products having XhoI/EcoRI restriction sites at their ends and COX2-specific stem sequences with 19bp loop helped to create miR30-adapted shRNAs. To recognise appropriate shRNAs, each clonig template was ligated into LMP, a retroviral vector with miRNA-based shRNA expression was driven from viral 5LTR promotor. To examined the suppression of cox-2 by shRNAs , NIH3T cells were transduced with 4 LMP vectors at very low multiplicity of infection. Southern blot method confirmed the integration sites in retrovirus transduced cell population with selected antibiotic. The examination of 4 cox2 specific shRNAs ability to block COX-2 protein expression, NIH3T3 cells were stimulated in serum medium and extracts for COX-2 protein content. These shRNAs did not have significant effect on cox-2 , on the other hand, cells transduced with cox-2 shRNAs 3 and 4 expressed nominal amount of cox-2 protein in response to serum stimulation. RAW264.7 cells transduced with luciferase shRNA vector expressed noticeable cox-2 protein response to bacterial lipopolysaccharide with compared to saline-treated cells.

With the western blot method, obtained the result that over 90% reduction of LPS-induced cox-2 expression seen. To examine the reason of inactivation of cox2 transcript by cox2.2058, prostaglandin E2 production in serum stimulated NIH3T3 cells expression observed. In a knock-in mouse, cox-2 firefly luciferase is expressed from endogenous cox2 gene and used this mouse to monitor expression from cox2 gene in variety of contexts. The classic inflammation model of Zymosan-induced paw inflammation is accompanied by a robust, self-resolving cox-2 induction measured by repeated non-invasive optical imaging in mouse. The paw inflammation model also very helpful to initiate triple transgenic shcox2/C3/Luc+ mouse. GFP fluorescence from paws of mice received DOX-containing diet was greater 10-fold increase compared to paws of mice did not receive DOX, shows that shcox2 expression was strongly induced by DOX.

(Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP)

The low dose of zymosan triggers a mild and transient inflammation resulted in full recovery, where higher dose of Zymosan resulted into more progreaaive and prolonged response leading to systemic inflammation. Isolated the cells from both models and quantified with cell counts determined by FACS analysis the throughout response. The determination of macrophage trafficking in resolving inflammation, influx of monocytes in zymosan response and mark the cell surface in both models. In confirmatory determination, seen that PKH26-PCL is cleared within 1 hour by cell-free inflammatory incubation from injected animal with PKH26-PCL macrophages and noticed that cultured macrophages did not label positively with PKH26-PCL. In this experiment, scientist found that M1 cells from proinflammatory model were more efficient compared to rM cells. The ability to kill bacteria of rM cells was enhanced by incubation with rp-cAMP inhibitor, whereas the same ability of M1 was reduced by addition of db-cAMP.

( Celecoxib Sensitizes S aureus to antibodies in Macrophages by Modulating SIRT1)

As the results, effect of combination of ampicillin and celecoxib on phagocytosed S aureus , its indicated positive results as reduction of bacterial growth compared to NASID drugs effect. The expression of SIRT1 at RNA and protein levels increased as a result when treated with celecoxib. SIRT1 increases the activity of antioxidant enzyme and peroxidase. Celecoxib-induced SIRT1 shows the decreasing expression of TLR2 upon flowcytometry analysis.

The reduction in cox-2 protein levels seen in the presence of SIRT1 gene. Activated SIRT1 gene also inhibited the expression of proinflammatory cytokine. Direct targets of SIRT1 were analysed by Western blot method, that suggested the activation of SIRT1 by celecoxib.

CONCLUSION:

With the experiments of above, it can be concluded that, inflammation is the very complex process and have some side effects which may results in death. But with the new drugs and these processes it can be stop.

There are some questions arises from these papers that unanswered till today:

1:COX-2 expressed in sterile inflammation and induced by pathogens but its role in anti-pathogens immune response is still not recognised.

2:At what time and in which condition, cox-2 expression is required to promote or supress infection and other biological process is not clear till now.

3:Which substances are trigger the resolution and restoration of tissue homeostasis like soluble mediators and cellular players.

highest distinction.

BS7101: Advanced Technique In Bioscience - Inflammation Is A Complex Physiological Response - University of East London - Nursing Assessment Answer, Download the solution from our nursing assessment expert.