key: cord-0901110-1hehw6qq
authors: Jeyaraman, Madhan; Muthu, Sathish; Bapat, Asawari; Jain, Rashmi; ES, Sushmitha; Gulati, Arun; Channaiah Anudeep, Talagavadi; Dilip, Shirodkar Jaswandi; Jha, Niraj Kumar; Kumar, Dhruv; Kesari, Kavindra Kumar; Ojha, Shreesh; Dholpuria, Sunny; Gupta, Gaurav; Dureja, Harish; Chellappan, Dinesh Kumar; Singh, Sachin Kumar; Dua, Kamal; Jha, Saurabh Kumar
title: Bracing NK cell based therapy to relegate pulmonary inflammation in COVID-19
date: 2021-07-21
journal: Heliyon
DOI: 10.1016/j.heliyon.2021.e07635
sha: 90aa3fd32707d2d97ea7b626b8d973d6c9aa5975
doc_id: 901110
cord_uid: 1hehw6qq

The contagiosity of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has startled mankind and has brought our lives to a standstill. The treatment focused mainly on repurposed immunomodulatory and antiviral agents along with the availability of a few vaccines for prophylaxis to vanquish COVID-19. This seemingly mandates a deeper understanding of the disease pathogenesis. This necessitates a plausible extrapolation of cell-based therapy to COVID-19 and is regarded equivalently significant. Recently, correlative pieces of clinical evidence reported a robust decline in lymphocyte count in severe COVID-19 patients that suggest dysregulated immune responses as a key element contributing to the pathophysiological alterations. The large granular lymphocytes also known as natural killer (NK) cells play a heterogeneous role in biological functioning wherein their frontline action defends the body against a wide array of infections and tumors. They prominently play a critical role in viral clearance and executing immuno-modulatory activities. Accumulated clinical evidence demonstrate a decrease in the number of NK cells in circulation with or without phenotypical exhaustion. These plausibly contribute to the progression of pulmonary inflammation in COVID-19 pneumonia and result in acute lung injury. In this review, we have outlined the present understanding of the immunological response of NK cells in COVID-19 infection. We have also discussed the possible use of these powerful biological cells as a therapeutic agent in view of preventing immunological harms of SARS-CoV-2 and the current challenges in advocating NK cell therapy for the same.

or LIR (immunoglobulin-like receptor) and Ly49 (homodimers) represent inhibitory 3 receptors [7] . Taking advantage of NK cell plasticity, improvement could be observed from 4 NK cell therapy through production of anti-inflammatory cytokine such as IL- 10. 5 This review article represents the fundamental role of NK Cells in the immuno-6 pathogenesis of COVID-19 and addresses the dire need for investigating the same rapidly 7 through the lens of prospective clinical trials in purview for solidly adducing its therapeutic 8 application in terms of efficacy and safety in COVID-19 patients respectively. 9 10 2. Anti-viral immunology of NK cells 11 12 NK cells produce and respond to inflammatory stimuli and play a role in anti-viral and tumor 13 immunology [11, 12] . The starring facets of these cells include the ability to sense RNA 14 viruses, critically responding to those viral invaders via optimal bridging of the innate and 15

adaptive immune system, and execute effector functions to escalate the process of viral 16 clearance. Upon stimulation, NK cells can produce antimicrobial and immuno-regulatory 17 cytokines. After an encounter of microbial challenges, innate cytokines (cells of the innate 18 immune system) elicit responses mediated by NK cell populations. Along with these innate 19 responses, NK cells promote immuno-regulatory functions by the down-streaming adaptive 20 response for defense against microbial organisms [13] . 

The interaction and cross-talk between viruses and NK cells have been well documented in 7 the literature. Viruses enter the host cells through a specific receptor binding mechanism 8 (direct fusion at the plasma membrane, or clathrin-or caveolin-dependent endocytosis of the 9 viral proteins) [16] . Viral proteins enhance immunogenicity through cell-cell interaction and 10 produce the host viral response. In absence of any specific viral receptors, non-specific viral 11 binding leads to internalization [17] . Though NK cells possess various receptors and ligands 12

for the viral protein entry, NK cells do acquire entry mechanisms through their direct contact 13 (involves immunological synapse) or by exosomal transfer from the virus-infected cells [18] . 14 The virus entry mechanism and NK cell modulation have been tabularized in Table 1 

A good insight into the pathogenesis of novel coronavirus disease (COVID-19) is necessary 5 to command over its management. The correlated evidence from severe patients with lower 6 lymphocytic counts highlighted how dysregulated immune system augments the 7 pathophysiological dynamics in COVID-19 patients [30] . The interaction of NK cells with 1 SARS-CoV-2 is shown in Figure 2 . 2

The deterioration of the respiratory system in COVID-19 is caused by a particular 3 exemplary dysfunction of the immune system. This is clearly evident from an unanticipated 4 deterioration of the patient just within 7-8 days after the symptoms start showing up. A study 5 included 54 COVID-19 patients, out of which 28 had a severe respiratory compromise, all 6 patients with respiratory compromise showed very low expression of HLA-DR and 7 macrophage activation syndrome (MAS). A heavy downfall in the count of CD8+ 8 lymphocytes, natural killer cells as well as CD19 lymphocytes was seen which clearly 9

indicates dysregulation of the immune system among these patients [30] . The decrease in the 10 In most of the COVID-19 patients, the symptoms ranged from mild to moderate, but 19 around 15% exhibited a progression to severe pneumonia and adding to the severity, a 5% 20 advanced to acute respiratory distress syndrome, MODS (multiple organ dysfunction 1 syndrome), and septic shock [31, 32] . Among patients associated with severe COVID-19 2 disease, a reduced number of CD8+ T cells, CD4+ T cells as well as NK cells and B cells 3 were seen, thus making lymphopenia a common finding [31] [32] [33] [34] [35] . Also, there was a drop in 4 the fraction of basophils, monocytes, and eosinophils [33, 36] . It has been reported that the 5 COVID-19 was more likely to occur in older men with comorbidities [31, [37] [38] [39] . 6

As depicted in Figure 2 , NK cells act as a virus responder in COVID-19 patients 7 without any co-morbidity as well as low-risk individuals but in high-risk individuals, NK cell 8 dysfunction supervenes and hence cytokine storm occurs, which may lead to ARDS and acute 9 lung injury. In high-risk individuals, the evasion of viral load fails as NK cells are 10 dysfunctional due to increased mononuclear cell recruitment besides the production of 11 inflammatory cytokines and chemokines as shown in Figure 3 . The first-line defense against a viral infection is an appropriately functioning innate immune 19 response, but when the same immune response is dysfunctional, it can lead to exaggerated 1 inflammation to which a patient may succumb to death [41] . 2

The two types of immunity in our body i.e. innate and adaptive; both these work 3 inconjunct to counteract the invasion of the pathogen in our body. Physical and epithelial 4 barriers, dendritic cells, phagocytes, and natural killer cells form the main constituents of the 5 innate immune system [42] . Notably, NK cells constitute one of the most important parts of 6 the innate immune system wherein their effector function does not need any pre-stimulation 7

[43]. 8 Natural killer cells are the front players in defencing immunologically against viral 9

infections and cancer through their cytolytic activity and production of cytokines [44-47]. NK 10 cells can respond to inflammation and recognize these molecular cues on certain target cells, 11 thereby facilitating the production of IFN-γ or the direct cytolysis of those target cells to 12 suppress the virus replication activity [13] . The potential source to isolate natural killer cells includes peripheral blood and secondary 7 lymphoid organs (lymph node, tonsil, spleen, and lymph) respectively. Notably, the 8 peripheral blood results in more quantities of NK cells in comparison to other lymphoid 9 organs [57]. The method of NK cell isolation from peripheral blood is described here. 10

By density centrifugation, lymphocytes are isolated from peripheral blood over a step 11 gradient consisting of a mixture of the carbohydrate polymer Ficoll and the dense iodine-12 containing compound metrizamide. The resultant comprises lymphocytes and monocytes. 13

Since these re-circulating lymphocytes are being isolated from blood, they never are a 14 representation of the lymphoid system [58] . The procedure of isolating the natural killer cells 15 has been discussed in 18 19 Collection of peripheral blood in an anticoagulant-containing tube and diluted with an equivalent volume of Phosphate Buffered Saline (PBS). ↓ Diluted blood (2/3 rd portion) is layered over 1/3 rd of Ficoll™ via pipette which results in the formation of interface distinctly ↓ Centrifugation for 30 mins at 800 g at room temperature resulting in the formation of a well-defined layer of lymphocyte at the interface ↓ Pipetting out the layer of lymphocytes from the interface into a fresh centrifuging tube with PBS dilution ↓ Centrifugation for 10 mins at 800 g at room temperature resulting in the formation of lymphocyte pellet (Refer Note) ↓ Resuspend the cell pellet in 40 μl of buffer per 10 7 total cells and add 10 μl of Biotin-Antibody Cocktail (human antibodies against antigens not expressed by NK cells) per 10 7 total cells ↓ Incubate for 10 min at 4°C and then wash with buffer by adding 10-20× labeling volume It is astounding that molecular mechanism is endowed with defective antigen 5 presentation and lymphopenia in combination whereby subjecting lymphoid cells to function 6 in a defective manner. At the same time, it is important to note that these monocytes serve as 7 potent cells for the assembly of TNF-α and IL-6 in severe respiratory failure (SRF) 8 exacerbated by SARS-CoV-2. The analysis of patients infected by SARS-CoV-2 showed 9 circulating concentrations of TNF-α, INF-γ, IL-6, and CRP respectively. INF-γ was below 10 the limit of detection, which indicates that the Th1 response does not involve inflammation. 11

There was a difference in the concentration of circulating levels of TNF-α among COVID- 19 12 patients. In contrast, the concentrations of IL-6 and CRP were significantly elevated in inflammatory cytokines including IFN α & γ and IL-1β also play a role in the cytokines storm 27 [95] . The interplay between the protective function of immune mediators and the striking 28 storm caused by the same mediators will decide the outcome. 29 Research to date provides us with a potential understanding of the biology of NK cells 25 concerning its function and its diversified interactions with receptors. Its role is extremely 26 well-substantiated in neoplastic conditions but warrants a clearer picture in the case of 27 autoimmune conditions and viral infections. The race of finding a definitive cure for COVID-28 19 has bought in striking efforts from the medical fraternity and researchers. The emerging 29 evidence in COVID-19 hints towards the involvement of NK cells in immune dysregulation 30 especially in severely ill patients. Still, there is a lack of understanding regarding the role of 31 NK cells in asymptomatic or early cases due to the inability of establishing the diagnosis in 32 clinics, and thereby the opportunity to collect their sample for research purposes is 33 undoubtedly skipped. Moreover, it is imperative to decide upon NK cell therapy will 34 J o u r n a l P r e -p r o o f perquisite by boosting (as in early presentation) or tuning (late presentation) respectively. 1 NK cell-based therapy may emerge as a major player provided investigations are accelerated 2 in this regard by overcoming this paucity. The current focus should be on establishing this 3 novel therapy wherein techniques for isolation and expansion of these cells in the required 4 count needs to be further addressed. 

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The role 1 of natural killer cells in resistance to the intracellular bacterium Listeria monocytogenesin 2 rats

Ribavirin improves the IFN-gamma response of natural killer cells to IFN-based 5 therapy of hepatitis C virus infection

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We thank Dr. Prajwal GS, Junior Resident of Orthopedics, JJM Medical 7