Immunoreceptors modulate eosinophilic functions in viral immunity

Abstract

Immunity a term used as resistance to pathogens, also referred to reactions of the body to noninfectious compounds such as tumors, harmless environmental substances and even sometimes host's components, each of which are called as tumor immunity, allergy and autoimmunity, respectively. The whole components like cells, tissues and molecules that generate this immunity is defined as the immune system and the immune response is coordinated by these components to foreign substances. Providing protection against infections or eradication of the infectious compounds from the body is the major physiological function of the immune system. Besides, growth of some tumors and cancers can be inhibited by stimulating immune reactions against cancer cells. The human defense system against pathogens can be divided into 3 levels: physical and chemical barriers, innate immunity and adaptive immunity. The innate immunity relies on a finite number of receptors for detecting the invading pathogens. These limited number of receptors target large groups of pathogens by recognizing conserved microbial patterns. Furthermore, activation of adaptive immune response is achieved by innate immune response. Innate immune cells include both hematopoietic and nonhematopoietic origin which make them different from adaptive immunity which relies on T and B lymphocytes. Innate immune response development involves hematopoietic cells that differentiate into monocytes, macrophages, mast cells, dendritic cells, natural killer cells, natural killer T cells, basophils, neutrophils and eosinophils. Moreover, hematopoietic cells include skin and epithelial cells reside in respiratory, genitourinary and gastrointestinal tracts. Innate immunity depends a few germline encodedreceptors which sense pathogen specific structural motifs. These receptors are collectively called pattern recognition receptors (PRRs) and microbial components recognized by PRRs are called pathogen associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PRRs can exist in various cellular compartments like cell membrane and endosomal membranes, or cytosol. Moreover, they can be found in extracellular environment such as bloodstream or interstitial fluids. They are mainly divided into four major types: 1) Toll-like receptors (TLRs), 2) Nucleotide binding and oligomerization domain-like receptors (NLRs), 3) Retinoic acid-inducible gene 1-like receptors (RLRs), 4) C-type lectin receptors (CLRs). Activation of these receptors of the innate immune cells result with induction of adaptive immune cells. Hence, PRRs help to generate immune response to eradicate infectious agents, for example they induce the death of the infected cells. Humans are constantly under the threat ofinvasive opportunistic microorganisms including viruses, fungi, bacteria and parasites. The detection and development of an appropriate immune response against invading viruses are crucial for the outcome of virus infections. Recognizing viral nucleic acids is the first step for sensing virus infection. This recognition mainly depends on the genetically predetermined and germline encoded PRRs. There are TLR protein family members recognize viral genetic material such as TLR3, TLR7, TLR8 and TLR9. Upon recognition of viral nucleic acids or proteins by PRRs, production of type I IFN is induced resulting in the activation of target cells in both autocrine and paracrine manners. TLR7 and TLR8 have the ability to detect the existence of various single stranded RNA (ssRNA) viruses. These viruses include influenza, vesicular stomatitis virus (VSV), HIV, Sendai virus (SV) and a number of coronaviruses and flaviviruses. TLR3 is an endosomal nucleotide sensor which is located on endosomes and activated by double stranded RNA (dsRNA). As dsRNA is the genome for many viruses or they synthesize dsRNA during their life cycles, TLR3 also detects the existence of dsRNA and DNA viruses. Unlike TLRs, RLRs are RNA detectors locolized in the cytosol. RLR protein family includes three members: RIG-I, MDA5 and LGP2. Upon viral RNA association and oligomerization, RLRs bind to mitochondrial antiviral signaling protein (MAVS) through its CARD. MAVS has an essential role in RLR signal transduction as an adaptor protein whichinduces TBK1 and IKKε leading to the activation of IRF3 and IRF7. These transcription factors and NF-κB, together, activate the production of type 1 IFNs. Moreover, NLRs are important part of the cytosolic innate immunity and have a role in various key pathways such as inflammasome, MAPK, NF-κB and type 1 IFN signaling. Activation of inflammasome complexes is essential because it induces inflammation by leading to the production and secretion of IL1β and IL18 also known as inflammasome dependent cytokines. Eosinophils attract considerable attention with their identified roles in many pathological processes such as acute and chronic infections, cancer and thrombosis. Although their accepted roles in parasitic infections, involvement of eosinophils in fungal, bacterial and viral infections is an on-going research topic in the field of immunology. These granulocytes contain and generate substances with antiviral functions such as RNases and they may also involved in adaptive immunity with their potential antigen-presenting ability. Together, findings about eosinophils indicate potential antiviral role for eosinophils which need to be explored further. Despite the studies on animal models and primary human eosinophils demonstrating the importance of eosinophils against viral infections, the question of how eosinophilic functions are regulated following he viral infections is still ambiguous. Thus, the aim of the study is to investigate the changes in eosinophilic functions upon activation of TLR3, TLR7 and TLR8 with proper ligands, poly(I:C), R848, and ssRNA40, respectively. low number of eosinophils in blood (1-6%) makes them difficult to study in vitro. Therefore, we utilized EoL-1 human eosinophil line as a model in our study which was previously shown to serve an ideal model due to the expression of eosinophilic markers In this study we initially determined the activation of Eol-1 cells upon treatment with viral ligands, eosinophilic PRRs and immune receptors. Secondly, surface markers of eosinophils were determined to further understand the eosinophilic functions. Also, cytokines that they released were analyzed to understand the potential involvement in the induction of adaptive immunity. Moreover, production of matrix metalloproteinases was measured. We observed significant increase in IL-6 and IFN-γ secretion upon TLR8 activation. Also, the number of IL5Rα and PDL1 expressing Eol-1 cells were augmented with TLR3, TLR7/8 and TLR8 inductions. Our data suggested roles for TLR3, TLR7 and TLR8 in eosinophilic functions. We then investigated the changes in granule content of eosinophils during viral infections. We showed that ECP mRNA levels were upregulated upon the ssRNA40 treatment in Eol-1 cells. This data also, demonstrate the possible roles of ECP on the regulation of antiviral eosinophilic functions. MMPs are matrix-degrading enzymes which have roles in leukocyte recruitment to chemokines during microbial infections. In addition to their functions in immune regulation, MMPs can also lead to tissue damage as a result of persistant pathogen infections or spread. Furthermore, CD147 is a matrix metalloproteinase inducer that plays critical roles in various viral infections. Recently, CD147 was shown as an alternative receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Therefore, we also investigated the MMPs and MMP inducer CD147to better understand their roles in eosinophilic antiviral immune responses. The results from RT-qPCR showed a relationship between both CD147 and TLR activation as well as the data obtained from gelatin zymography clearly indicated the relationship between MMP-9 and TLR activation in Eol-1 cells. All stimulants tested in this study elevatedthe CD147 at mRNA level in Eol-1 cells. Additionally, TLR3 and TLR8 ligands significantly increased the Pro-MMP9 production at 24 hours post-induction in EoL-1 cells. Upregulation of TLR8 led us investigate the potential adaptor molecules which may have roles in downstream signaling pathways through TLRs in eosinophils. CARD9 is a multifunctional adaptor protein which participates in different phases of the immune system including fungal, bacterial and viral infections. Thus, we investigated the CARD9 expression in Eol-1 cells upon TLR3, TLR7/8 and TLR8 inductions. Despite the decreased mRNA expressions of CARD9, there was a significant increase in CARD9 expression at protein level in Eol-1 cells. Next, we measured the expression levels of cytosolic PRR RIG-I at protein level because of its well-known roles in antiviral immunity. We observed 1.49-fold increase in the cells stimulated with TLR8 agonist (ssRNA40, 2 μg/ml). Then, we measured NOD2 as it is already known as the interaction partner for CARD9. Numerous studies formerly showed the NOD2 involvement in response to many RNA virus infections such as parainfluenza virus, VSV, RSV and IAV. Here we showed that NOD2 protein expression was significantly increased after induction with TLR7/8 and TLR8 ligands in Eol-1 cells. Overall, of all the stimuli we tested, ssRNA40 (TLR8 signaling) was the most potent in inducing the mRNA expressions of ECP and CD147, and protein expression of immune receptors, surface markers of eosinophils and cytokines. Interestingly, CARD9 which is a critical adaptor against fungi infections was significantly increased after induction with TLR7/8 ligands, suggesting an important role for TLR7 and TLR8 rather than TLR3 in antiviral immune response generated by eosinophils. Our findings will pave the way for future studies focusing on eosinophil related infectious diseases.