Role of Chemokine Receptor, CXCR4 Mediated Signaling in Cellular Senescence

Cellular senescence has been proposed to be equivalent to organismal aging and is one of the outcomes of the cell fate decision process in response to DNA damage that occurs in cells. When a cell encounters DNA damage, the cell cycle is immediately halted to evaluate which decision to take in response to genomic insult. The choices are between repairing the damage and continue division, or enter a non-replicative but viable state called senescence or to die if damage is severe (Figure 1). The signaling cascade, which detects this damage and regulates the cell fate decision, is collectively called as DNA damage response (DDR). However, the exact mechanism of how delineation for each decision happens is still not clear. Since DNA damage works as a mediator for cell fate decision, my work aimed to study senescence as a DNA damage response. In addition, the role of free radicals like ROS in cellular senescence is not very clear because though an increase in their concentrations is recorded in aged cells, it is not evident if the increase seen the cause or the effect of aging, primarily because they themselves capable of causing DNA damage. This conundrum have always led to confounding observations wrt role of free radicals in the cellular senescence process and if the senescence is caused through agents which rely on ROS to cause DNA damage, ROS becomes absolutely integral to the aging process. To understand this aspect formed the first line of investigation in my work along with identification of the sensor of DNA damage, which drives various cell fates. During organismal ageing there is an accumulation of senescent cells, which could be the major reason for functional decline of tissues and organs with age. However, to study changes associated with signaling molecules with respect to ageing, a cellular model system for senescence driven through DNA damage was needed, using which interplay between senescent / aged cells and cellular niche can be established. Studying the spatial and temporal alterations in signaling dynamics, within the cell as well as with the neighbouring niche during the senescence process in anticipated to provide us better understanding about the complex process of ageing. For this, the objectives were defined to establish and characterize the DNA damage induced senescence model using various parameters, and especially study the signaling dynamics of GPCR mediated signaling in senescence. The role of chemokine receptor, CXCR4 and its ligand, CXCL12 mediated signaling was chosen for the study. The following sections describe the findings that were obtained from the various objectives studied during the course of this study. Section 1. Development and characterization a model system to study cellular senescence as a DNA damage response. In this part of the study, I characterized genotoxic stress induced cellular senescence model using 5-Bromodeoxyrudine as the DNA damaging agent. BrdU, owing to its property of being a thymidine analogue, is incorporated in dividing cells, and this incorporation is recognized as DNA damage. This triggers ‘persistent’ DNA damage response signaling, including activation of ATM kinase, one of the primary DNA damage sensor. As anticipated, the DDR response detected was directly proportional to the dose of BrdU treatment and so was Reactive Oxygen Species (ROS) levels, a known senescence mediator. Using this model system of direct DNA damage mediated DDR activation and induction of cellular senescence, the growth-arrested cells were extensively characterized for presence and quantum of most of the senescence associated markers known in literature. BrdU treated cells, which became senescent showed presence of DNA damage, morphological changes like flat, enlarged, granule rich appearance, expression of senescence associated molecular markers like p21, IL8, showed senescence associated beta galactosidase activity, refractiveness to growth factor for division, increased ROS levels, Golgi dispersion, etc. The secretome of the treated cells also showed increased secretion of inflammatory cytokines which are attributed to a senescence phenotype, called as Senescence Associated Secretory Phenotype (SASP), which triggered proliferative and migratory effect on cancer cells. Overall, in this part of the study, it was established that BrdU can cause DNA damage and induce senescence as one of the cell fate in response to the intermediate dose of damage. The senescent cells generated in the model system was established to be akin to senescence observed by replicative exhaustion of normal cells, thereby making our model applicable to the physiological studies as well. Section 2. Insights into the role of ATM-ROS axis during senescence initiation and maintenance using DDR mediated cellular senescence model. While the BrdU model system for generating senescent cells was being developed and characterized, it was observed that there is an increase in ATM activation as well as ROS production concomitant to the a dose of BrdU. At the same time it was also observed that senescent cells showed persistent DDR signaling and high levels of ROS. Using this premise, in the second objective of my study I aimed to identify if ATM and ROS are critical during initiation of senescence, when the cells are insulted with the DNA damaging agent or during the maintenance of senescent state of the cells. By quenching ROS during the initiation state, I recorded that ROS is not critical for inducing senescence and perhaps the increase in ROS levels in senescent cells is due to their higher metabolic activity. By inhibiting ATM activation during DNA damage, it was observed that BrdU induces senescence through direct DNA damage, and active ATM and DDR signaling is absolutely critical for the senescence initiation. It was also established that ATM is not just a DNA damage sensor but also a redox regulator in the senescence model system. Prevention of ATM activation in presence of DNA damage blocked senescence initiation and also triggered increased ROS levels in the cells affecting their long term viability, suggesting ATM regulates ROS levels as well in addition to sensing DNA damage. In order to study the role of ATM-ROS axis in the maintenance of senescence state, already senescent cells were subjected to ROS quenching and/ or ATM inhibition and it was identified that both these signaling molecules are essential for maintaining the viability of senescent cells. The findings from these study thereby show that senescence can be divided into two temporally distinct stages, initiation or early senescence stage and second, maintenance stage of senescence. Overall, I was able to characterize the presence of temporally linked ROS – dependent and ROS – independent events in cellular senescence, which are independently mediated by ATM kinase (Figure 1). Dose of Genotoxic Stress damage DDR Senescence initiation Repair Cell cycle ATM arrest kinase Death Growth arrest Senescence maintenance Senescence Cell ROS viability Elevated metabolism Figure 1. Signaling cascades regulating senescence onset and maintenance mediated through DDR. Cells enter senescence state in response to DNA damage, depending on the dose of insult, through an ATM dependent and ROS independent pathway. Unlike this ATM-ROS axis is critical for the maintenance of senescent state of damaged but viable cells. Section 3. Understanding the role of CXCR4 – CXCL12 mediated signaling in senescence. Age dependent changes in cellular signaling are less explored and I was specifically interested in understanding how presence of senescent cells affects its microenvironment or vice versa i.e. how microenvironment affects senescent cells. In this premise the third objective of this study was defined towards identifying role of a GPCR, CXCR4 mediated signaling in cellular senescence and associated inflammation. CXCR4 is a ubiquitously expressed GPCR and it’s only known ligand is CXCL12/ SDF1 (stromal derived factor ), which is a homeostatic chemokine (i.e. its levels does not change under most physiological conditions). During characterization of DNA damage induced senescence model system, it was observed that this receptor expression is induced during DNA damage ells, which was also found to be so from data available from other gene expression studies as well. During the course of my work, I identified that senescent cells show CXCR4 up regulation in response to DNA damage, mediated through activation of ATM kinase - HIF1 axis and plays a critical role in enhancing the senescence associated inflammatory response in presence of its ligand, CXCL12. This CXCL12 dependent enhanced inflammatory response in damaged cells was determined to be sensitive to the pertussis toxin treatment and hence dependent on G protein activation. Further downstream analysis revealed the pro-inflammatory effect of the CXCR4 receptor activation was due to cAMP level suppression post activation by the Gi subunit. Given that cAMP levels are antagonistic to inflammatory phenotype, using a library of pharmacological compound library, I also discovered that cAMP specific PDE, phophodiesterase 4A, is also involved in regulating inflammatory response during the initiation stage of cellular senescence. The screen also confirmed the involvement of previously identified molecular components such as p38 MAPK and leukotrienes in the senescence associated inflammatory phenotype. The examination of the role of the CXCR4- CXCL12 axis in the deeply senescent cells surprisingly revealed that deeply senescent cells are refractory to CXCL12 stimulation in terms of inflammatory response, which was experimentally determined to be associated with impaired calcium release. Overall, the findings from this part of the study revealed a novel signaling cascade where CXCR4 up regulation is a part of the DDR response in cells, which utilizes the Local Excitation Global Inhibition (LEGI) mechanism to enhance the sensitivity of the damaged cells to its ligand CXCL12. This enhanced sensitivity mediates the CXCL12 dependent inflammatory response, which aids in attracting immune cells for clearance of these damaged cells. Once the cells have entered the senescent state, the axis is physiologically down modulated and the senescent cells showed refractiveness to CXCL12 stimulation, probably to prevent persistent acute inflammation, if the senescent cells are not cleared (Figure 2). Figure 2. CXCL12-CXCR4 axis in cellular senescence. During senescence initiation stage, when cells encounter DNA damage (Step 1), there is induction of CXCR4 receptor (Step 2), which enhances of CXCL12 mediated signaling for increased inflammatory response (Step 3). In the maintenance stage, where the cells are not cleared (Step 4), the axis is suppressed (Step 4), thereby bringing the levels of inflammatory secretome down, and thereby preventing damage to the cells (Step 5)

The possible role of soluble material from macrophages in cell mediated immunity in pulmnary tuberuculosis

Lymphocytes from pulmonary tuberculosis patients and healthy controls showed identical proliferative responses to mitogen (PHA) and antigen (PPD) on Day ‘O’ (DO), Day-2 (D2) and Day-7 (D7) of culture. Also, there was no suppression of PHA induced lymphocyte proliferation in the presence of culture supernatants of pulmonary tuberculosis patient’s mononuclear cells

Immunology of tuberculosis - An overview

Over a hundred years have passed since Robert Koch has isolated the tubercle bacillus, but still the immune mechanisms in tuberculosis are not yet completely unravelled. The information explosion in the field of immunology during the last ten years has resulted in a modest beginning in the knowledge of tuberculoimmunity at the cellular and molecular level. It is necessary to understand the host responses to mycobacterium tuberculosis in order to appreciate the clinical consequences, diagnosis and prophylaxis of tuberculous infection. In the following sections the progress in our understanding of host’s immune response to M. tuberculosis is considered with special emphasis on immunopathogenesis, immunosuppression and immunodiagnosis

Hydrogen peroxide release by OKI A1 (anti DR-Monoclonal antibody) resustabt alveolar mnacrophages in tuberculosis

Phorbol myristate acetate (PMA) triggered hydrogen peroxide (H2O2) release from alveolar macrophages and corresponding blood monocytes were studied as a whole, in active tuberculosis, inactive tuberculosis (treated), non-tuberculous lung disease patients and normal individuals. Irrespective of the study subjects, the alveolar macrophages produced less H2O2 than the corresponding blood monocytes. The alveolar macrophages that were resistant to OKIa1 (Anti-DR monoclonal antibody and complement treatment) produced an increased level of H2O2 than the control ascites and complement treated alveolar macrophages. Moreover, such increase in H2O2 release was not seen with peripheral blood monocytes; more than 90% monocytes were OKIa1 resistant population. These OKIa1 resistant alveolar macrophages are probably important in their metabolic, microbicidal and the immunological functions

Hydrogen peroxide producing potential of alveolar macrophages & blood monocytes in pulmonary tuberculosis

Alveolar macrophages from patients with active pulmonary tuberculosis, inactive tuberculosis (treated patients), non-tuberculous lung disease and from normal healthy individuals were tested for their ability to produce hydrogen peroxide (H2O2) upon stimulation with phorbol myristate acetate (PMA) in vitro. PMA induced H2O2 production was significantly higher in all the groups of patients studied when compared to the normal subjects. Among the four groups studied, the spontaneous release of H2O2 was increased in the alveolar macrophages of smokers than non-smokers. There was no difference in the spontaneous and PMA induced H2O2 production between the non-smoker group of the active tuberculosis patients and the normal non-smoker group. Further, the blood monocytes of the activepulmonary tuberculosis patients and those of normal controls were equally competent in producing H2O2, in vitro, upon stimulation with PMA. The study suggests that the increased production of hydrogen peroxide by alveolar macrophages is not specific for tuberculosis

Identification of a 3-Alkylpyridinium Compound from the Red Sea Sponge Amphimedon chloros with In Vitro Inhibitory Activity against the West Nile Virus NS3 Protease.

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 μg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication

Immunological investigations in tuberculous ascites

Cell mediated immunity was assessed in seven patients with bacteriologically and/or histologically confirmed tuberculous ascites. Eight non-tuberculous ascites patients were included as controls. Anti-PPD antibody levels were also estimated by ELISA. Macrophage from tuberculous ascitic fluid showed increased production of H202 when compared with ascitic fluid macrophages from controls. Proliferative response of lymphocytes to PPD antigen was greater in ascitic fluid than in peripheral blood in tuberculous patients, while the responses were reversed in control patients. Tuberculous ascitic fluid had higher levels of anti-PPD antibodies than ascitic fluid from controls, though their levels in peripheral blood were similar in the two groups. It is concluded that the results provide support to the concept of immunologic localization

Assessing the Wastewater Pollutants Retaining for a Soil Aquifer Treatment using Batch Column Experiments

In this study, the Secondary Treated Waste-Water (STWW) to infiltrate through the soil matrix, hence eliminating the contaminants in the effluent. For this study, three types of soil, such as loamy sand, fine sand, and clayey soil, were subjected to two cycles of wetting and drying to assess the type of soil that removes the maximum contaminants under which cycle. At diverse locations, soil samples were collected and examined to determine the soil types. Likewise, STWW was collected from Chennai Metropolitan Water Supply and Sewerage Board (CMWSSB) and Perungudi Sewage Treatment Plant (PSTP) to illustrate the quality of water before Soil Aquifer Treatment (SAT). About 1.5 m in height and 8 mm in diameter of fabricated acrylic material columns are used for the soil aquifer treatment efficiency studies. Water quality parameters, namely pH, TDS, and turbidity, were monitored throughout the study. All the organic compounds present in water were reduced to a higher level only in the fine sand in the one-day wetting/drying cycle. pH was reduced from 8.55 to 7.5, TDS was reduced from 1580 mg/l to 850 mg/l, and Turbidity was reduced from 7.24 to 4.04 NTU. This proposed method is to minimize the amount of water pollution from the environment. It is an effective way to manage aquifer recharge (MAR). SAT is the easiest method, aquifer and/or soil-based treatment systems improve the effluent quality of wastewater by removing the trace elements in the aquifer during the recharge of groundwater. It is likewise attractive for technologically advanced as well as emerging countries, and it is supple enough to allow adaptation to home-grown requirements by uniting it with predictable natural or bringing about water and technologies of wastewater treatment. Doi: 10.28991/CEJ-2022-08-07-011 Full Text: PD

Concurrent use of prescription drugs and herbal medicinal products in older adults: A systematic review

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The use of herbal medicinal products (HMPs) is common among older adults. However, little is known about concurrent use with prescription drugs as well as the potential interactions associated with such combinations. Objective Identify and evaluate the literature on concurrent prescription and HMPs use among older adults to assess prevalence, patterns, potential interactions and factors associated with this use. Methods Systematic searches in MEDLINE, PsycINFO, EMBASE, CINAHL, AMED, Web of Science and Cochrane from inception to May 2017 for studies reporting concurrent use of prescription medicines with HMPs in adults (≥65 years). Quality was assessed using the Joanna Briggs Institute checklists. The Evidence for Policy and Practice Information and Co-ordinating Centre (EPPI-Centre) three stage approach to mixed method research was used to synthesise data. Results Twenty-two studies were included. A definition of HMPs or what was considered HMP was frequently missing. Prevalence of concurrent use by older adults varied widely between 5.3% and 88.3%. Prescription medicines most combined with HMPs were antihypertensive drugs, beta blockers, diuretics, antihyperlipidemic agents, anticoagulants, analgesics, antihistamines, antidiabetics, antidepressants and statins. The HMPs most frequently used were: ginkgo, garlic, ginseng, St John’s wort, Echinacea, saw palmetto, evening primrose oil and ginger. Potential risks of bleeding due to use of ginkgo, garlic or ginseng with aspirin or warfarin was the most reported herb-drug interaction. Some data suggests being female, a lower household income and less than high school education were associated with concurrent use. Conclusion Prevalence of concurrent prescription drugs and HMPs use among older adults is substantial and potential interactions have been reported. Knowledge of the extent and manner in which older adults combine prescription drugs will aid healthcare professionals can appropriately identify and manage patients at risk.Peer reviewedFinal Published versio