Exploring the modulation of immune response and oxidative stress of intracellular pathogens using nanoparticles encapsulating drugs

Ramendra Pati Pandey, Anjali Priyadarshini, Archana Gupta, Arpana Vibhuti, Elcio Leal, Utpal Sengupta,Vishwa Mohan Katoch, Pawan Sharma, Catrin E Moore,V. Samuel Raj Centre for Drug Design Discovery and Development (C4D), SRM University, DelhiNCR, Rajiv Gandhi Education City, Sonepat 131 029, Haryana, India; ramendra.pandey@gmail.com (R.P.P.); anjali.p@srmuniversity.ac.in (A.P.); archana.g@srmuniversity.ac.in (A.G.); arpana.v@srmuniversity.ac.in (A.V.); directorcd4@srmuniversity.ac.in (V.S.R.); Stanley Browne Research Laboratory, The Leprosy Mission, Nand Nagari, Sahadra, New Delhi, 110093; usengupta2002@yahoo.com (U.G.); Rajasthan University of Health Sciences (RUHS), Jaipur, President, JIPMER, Puducherry; vishwamohankatoch18@gmail.com (V.K.M); ICGEB (International Centre For Genetic Engineering And Biotechnology), New Delhi 110067, India; pawan37@gmail.com (P.S.) Institute of Biological Sciences, Federal University of Para, Para 66075-000, Brazil; elcioleal@gmail.com (É.L.) Nuffield Department of Medicine, University of Oxford, Big Data Institute, Li Ka Shing Centre for Health Information and Discovery Old Road Campus, Headington, Oxford, OX3 7LF, United Kingdom, catrin.moore@ndm.ox.ac.uk (C.M.) Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 October 2020 doi:10.20944/preprints202010.0580.v1


Abstract:
The immune system is a dynamic network of cells and cytokines are the major mediators of immune responses which combat pathogens. Based on the cytokine production, effector T cells differentiate into subsets known as Th1, Th2, Th17 or Treg (T regulatory). This system serves as a barrier to intracellular pathogens, bacterial infections and stimulates the production of reactive oxygen species (ROS), reactive nitrogen intermediates (RNI) and nitric oxide (NO), which diffuses across membranes and engulfs intracellular pathogens. Oxidative stress occurs when ROS, reactive nitrogen species (RNS) production and antioxidant defences become imbalanced. Oxidative stress generated by infected cells produces a substantial amount of free radicals which enables killing of intracellular pathogens. Intracellular pathogens are exposed to endogenous ROS as part of normal aerobic respiration, also aexogenous ROS and RNS are generated by the host immune system in response to infection. Nanoparticles which are designed for drug delivery are capable of trapping the desired drug in the particles which protects the drug from enzymatic degradation in a biological system. The small (subcellular) size of nanoparticles enables higher intracellular uptake of the drug which results in the reduction of the concentration of free drugs reducing their toxic effect. Research on the modulation of immune response and oxidative stress using nanoparticles used to encapsulate drugs has yet to be explored fully. In this review we illustrate the immune activation and generation of oxidative stress properties which are mediated by nanoparticle encapsulated drug delivery systems which can make the therapy more effective in case of diseases caused by intracellular pathogens.

Introduction:
The immune system is constantly in a flux, it encompasses a dynamic network of cells, tissues and organs within a host that work in a coordinated manner to defend the body against attacks by "foreign" invaders while also protecting against disease by recognising both "self" and "non-self". Antigens, usually a toxin or foreign substance recognised by the host, are recognized by specialized cells which facilitate their initial destruction followed by elimination from the host. Any microorganism able to cause disease in a host organism can be termed a pathogen. When a pathogen (for example a bacterium, virus or protozoal parasite) infects the human body, after which an internal battle ensues between the host's innate and adaptive immune system and the pathogen's assorted virulence mechanisms, together with factors which are able to overcome the immune attack and establish disease. Detection of antigens by the host is complicated as pathogens evolve rapidly; are able to adapt quickly and escape the immune surveillance which allows the pathogens to infect their hosts and cause disease (Christensen & Thomsen, 2009). Pathogens can be extracellular and intracellular and the mechanism to counter their attack by immune system is varied.
As intracellular pathogens reside within the host cell, their elimination and clearing is more complex, the cell-mediated immune response plays a vital role in the host defence against intracellular pathogens such as those causing tuberculosis and leishmaniasis (Urdahl, 2014) .
The immune response can be both innate and adaptive; the innate immune response is the first line or primary defence immediately stimulated upon infection. This first line of defence initiated by the host upon the entry of microorganisms to the body involves responses by phagocytic cells that fight pathogens in a nonspecific manner. Antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs), which are spread extensively throughout the body, swallow and process potential microbial antigens via phagocytosis, antigen presentation and activation of T and B lymphocytes to generate an adaptive immune response (Heit el al., 2008). These activated cells cooperate with activated macrophages within the host to abolish intra and extra-cellular pathogens (Storni et al, 2005). Antigens are collected by APCs after which they migrate to the draining lymph nodes with maturation signified by enhanced presentation of antigenic material to major histocompatibility complex (MHC) class I and and/or class II receptor molecules that are subsequently presented to the immune system for development of the acquired immune response. The

Immune evasion:
Many pathogens which have the ability to cause acute infections are often cleared effectively by the hosts immune system. However, some pathogens which invade the hosts cells become intracellular pathogens, and are able to establish persistent and sometimes lifelong infections. Several of these intracellular pathogens manage to evade the host immune system causing disease by replicating inside the host cells.
Some bacteria, such as M. tuberculosis are able to disrupt the phagosome-lysosome fusion using the PtpA tyrosine phosphatise which preventing the acidification of the phagosome (Bach et al., 2008). Other mechanisms use the phagosome to create a suitable microenvironment for proliferation: Legionella pneumophila safeguards itself from the hosts innate immunity by creating a vacuolar environment which is lacking MHC class II molecules (Clemens and Horwitz, 1992), whereas C. burnetii needs an acidic environment for growth and virulence which eliminates the other pathogens (Maurin et al., 1992). Some bacteria can enter a hardy, non-replicating T. brucei uses its "vector host" to its advantage, the saliva of the Tsetse fly is transmitted along with the parasite, the saliva contains a Gloss2 peptide which suppresses human host release of cytokines TNF-α, IFN-γ, IL-6, and IL-10 (Stijlemans et al., 2016). Helminths are able to survive in humans for many years due to their ability to secrete immunomodulatory products, including proteases, protease inhibitors, venom allergen homologues, glycolytic enzymes and lectins (Hewitson et al., 2009). As central components of the "respiratory burst" in activated macrophages and neutrophils, the reactive oxygen species (ROS) and reactive nitrogen species Glutathione-S-transferase (GST) are a family of Phase II detoxification enzymes catalyzing the detoxification of electrophilic compounds, it protects cells from mutagens and carcinogens as a free radical scavenger along with glutathione (Hemachand, 2002). Glutathione removes H2O2 in the cytosol (Reed,1969). H2O2 is formed near the membrane during phagocytosis, and it can simply diffuse into the cytosol. In the cytosol, glutathione reacts with H2O2 through a chemical reaction catalyzed by glutathione peroxidase which results in glutathione disulfide (GSSG) (Paul, 1970). Glutathione reductase (GR) catalyzes the regeneration of glutathione from GSSG, utilizing NADPH generated by the hexose monophosphate shunt (HMPS).
Glutathione Peroxidase (GPx) isoenzymes use GSH as a donor of reducing equivalents to detoxify H2O2, in various organic compounds (Briviba, 1998).  (Sies, 1995). The cellular pool of glutathione is replenished by two mechanisms: glutathione regeneration from GSSG mediated by GR and de novo glutathione synthesis. By regenerating glutathione from GSSG, GR facilitates cytosolic H2O2 detoxification, which protects phagocytes from oxidative damage and sustains oxidative burst-mediated bactericidal activities (Reed, 1969). In oxidative stress there is either an excessive production of ROS or a significant decrease or lack of antioxidant defense. The removal of H2O2 or other hydroperoxides by GPx requires GSH as cofactor. GSH is a tripeptide of glutamate, cysteine, and glycine, which is found ubiquitously in eukaryotic cells at a concentration between 1 and 10 mM. GSH has a potent electron-donating capacity. Its high redox potential reduces GSH which is both a potent antioxidant and a convenient cofactor for enzymatic reaction.
These molecules are all associated with the regulation of apoptosis (Riley et al., 2006). NO is a component of the innate immune system, and is involved in both the pathogenesis and control of several types of viral, bacterial and parasitic infections (Bogdan, 2001). Furthermore, NO is able to modulate the immune response via the regulation of apoptosis and the upregulation of cytokine mRNA expression (Hanum et al., 2003). The regulation of NO production in tuberculosis appears to be very complex, due to the ability of various mycobacterial cell wall components to stimulate the release of NO (Underhill, 1999). Basu  2) Penetrates the cell membrane using hydrophobic particles.
3) Nanoparticles <5 nm are transported across the cell membrane channel.
The nanoparticle structures allow a better retention of the drug inside the polymeric network and can slowly be degraded by esterase action.
There are two types of nanoparticles: a. Nanospheres (these have a solid framework) b. Nanocapsules (these have a liquid central cavity surrounded by a wall). Hyaluronic acid (Illum, 1994), Polysaccharides (Artursson, 1984), Chitosan (Verma, 2011;Artursson, 1984), have attracted considerable attention as potential drug delivery devices in view of their applications in the controlled release of drugs, their ability to target cells / tissues, and in their ability to deliver drugs in optimal dose at targeted sites (Langer, 2000). The synergistic effect of NPs when placed in combination rather than using a single NP and for combinations to target a broad spectrum of both Gram-positive and Gram-negative bacteria, could be useful to healthcare practitioners and biomedical engineers in the development of medical devices that target a wide range of bacterial pathogens (Bankier et al., 2019). FIGURE 2. Schematic representation of nanoparticles as a carrier system and its applications.

Conclusion:
Infectious diseases caused by pathogenic bacteria are one of the most common causes of death worldwide and are a constant health risk in all countries (Marova et al., 2011). Today, the burden of infectious disease on health, economy and other social aspects is so complex, that the worldwide cost cannot be estimated (El-banna et al., 2012). Multi-drug resistant (MDR) pathogens constitute one of the most severe worldwide public health problem. Apart from those, the biodegradable polymers can be utilized as drug delivery system because of several advantages such as biodegradability, biocompatibility, enhanced circulation and reduced toxicity. The main objective in using the nanoparticles as a delivery system is the control led release of encapsulated controlled drugs in order to accomplish the site specific action at the therapeutically optimal level. In addition, they can also influence the entrapment efficiency, release of the drugs from the nanoparticles and stability of nanoparticles. The area of modulation of immune response and oxidative stress using nanoparticles encapsulating drugs is yet unexplored. In this review we are trying to discuss the possible hypothesis whether the immune activating and generation of oxidative stress properties of nanoparticles encapsulated drugs delivery system can be co-related for progressing toward more effective therapy and immune response in case of infections caused by intracellular pathogens. Using antibacterial encapsulated nanoparticulate systems we may lead to a delay or inhibition of the resistance development. The shining ray of hope amidst the plethora of antimicrobial resistance is that the microbes would require multiple gene mutations in the same bacterial cell to become resistant to NPs. It will be interesting to explore further; an interplay of immune response, ROS and RNS created by intracellular pathogens, which warrant detailed evaluation on the signaling pathways to ascertain the extent of interdependence.