Poly I:C stimulation in-vitro as a marker for an antiviral response in different cell types generated from Buffalo (Bubalus bubalis)

Highlights

• Buffalo myeloid and non-myeloid cells recognize and respond differently to cytoplasmic delivered poly I:C.

• Compare to myeloid cell, non-myeloid cell found quicker in detecting poly I:C consequently, robust IFN-β and ISGs expression in fibroblast.

• High basal level expression of OAS1 and RNaseL in myeloid cells, may inversely influence the IFN-β expression in monocyte and macrophage.

• HMW poly I:C was better inducer of IFN-β in fibroblast, while LMW was favoured in monocytes and alveolar macrophage.

Abstract

The innate immune system is activated upon virus invasion of a host cell by recognizing viral component, such as dsRNA through specific receptors, resulting in the production of type- I IFNs, which confer an antiviral state within the invaded as well as surrounding cells. In the present study, fibroblast, monocyte and macrophage cells derived from water Buffalo (Bubalus bubalis) were exposed to a synthetic dsRNA analogue, poly I:C to mimic viral invasion in each cell type. Recognition of poly I:C through cytosolic helicase receptors RIG-I and MDA5 molecule lead to the activation of the RLR pathway, subsequently activating the MAVS-IRF3/7 cascade and the production of antiviral effector molecule like IFNβ and ISGs. Within the different cell types, we identified variability in RLR receptor and IFNβ expression after poly I:C administration. Fibroblasts responded quickly and strongly with IFNβ production, followed by macrophages and monocytes. Despite absolute expression variability among different cell types the expression trend of RLRs pathway genes were similar. Length of poly I:C molecule also influence IFNβ expression in response of RLR pathway. Short (LMW) poly I:C induce stronger IFN-β expression in myeloid (macrophage and monocyte) cells. In contrast long (HMW) poly I:C preferably elicit higher IFNβ expression in non-myeloid (fibroblast) cell. Therefore, MDA5 and RIG-1 plays an indispensable role in eliciting antiviral response in non- immune (fibroblast) host cell. Thus, stimulation of RLR pathway with suitable and potentially cell-type specific agonist molecules successfully elicit antiviral state in the host animal, with fibroblasts conferring a stronger antiviral state compared with the monocytes and macrophages.

Introduction

Indian cattle species (Bos indicus) and Buffalo (Bubalus bubalis) have been domesticated since 8000 yr and 5000 yr, respectively (Troy et al., 2001; Groeneveld et al., 2010), and have adopted to a wide range of climatic conditions of India. These geoclimatic conditions helped Indian cattle and buffalo to develop resistance against common infectious diseases over time in contrast to exotic cattle (Bos taurus). Indeed, various reports show that Indian zebu cattle and buffaloes are less susceptible to bacterial, viral and parasitic diseases (Ameni et al., 2007; Capparelli et al., 2007; Allen et al., 2010; Chaussepied et al., 2010), compared with Bos taurus breeds. Within these exogenous breeds, diseases such as Theileriosis, Anaplasmosis, Piroplasmosis, Trypanosomosis, Brucellosis, Haemorrhagic Septicaemia and rotavirus infection are more common (FAO, 1999; Glass and Jensen, 2007). Moreover, indigenous cattle have been reported to be less susceptible to infection with Mycobacterium bovis, compared to exogeneous and crossbred cattle (Finlay et al., 2012; Vordermeier et al., 2012). This non-specific disease resistance may be based on several factors such as proportions of peripheral γδ T cells (Telfer and Baldwin, 2015; Lawand, 2017), differences in the anti-viral response through type 1 interferons (IFN) (Katze et al., 2002), accessibility of cellular receptors for pathogen invasion, degree of apoptosis to restrict pathogen multiplication and dissemination (Hyama et al., 2003) and interplay of different cytokines to prevent pathogen multiplication in vivo.

To assess potential differences in anti-viral response synthetic polynucleotide, which mimic viral RNA such as poly I:C of different lengths, have previously been used to investigate the (innate) immune response in vitro (Kato, Takeuchi, Mikamo-Satoh, et al., 2008; Kato et al., 2011; Mian et al., 2013). Poly I:C is recognized by a redundant system of innate immune receptors, including the membrane-associated Toll Like Receptor (TLR) 3 as well as the cytosolic receptors Retinoic Acid-Inducible Gene I (RIG-I) and Melanoma Differentiation-Associated protein 5 (MDA5). TLR3 and RIG-I/MDA5 belong to the TLR and RIG-I Like Receptor (RLR) families of pathogen recognition receptors (PRRs), respectively (Kato et al., 2011). TLR3 and RIG-I/MDA5 interaction with either vesicular or cytoplasmic poly I:C lead to the activation of downstream molecules. Ligation of these PRR leads to activation of the adaptor proteins TIR-domain containing adapter-inducing interferon-β (TRIF) and Mitochondrial Anti-Viral Signalling (MAVS), respectively followed by the activation of cytosolic TNF Receptor Associated Factor 3 (TRAF3) and TANK Binding Kinase 1 (TBK-1) protein (Kato et al., 2011; Carneiro et al., 2017). This activation leads to the phosphorylation of Interferon Regulatory transcription Factor 3/7(IRF3/7), which form homodimers and are translocated into the nucleus. Here, they bind to a variety of promoter elements, thereby stimulating the expression of type I IFNs and proinflammatory cytokines.

IFNβ is a key molecule of class I interferon, and its expression is upregulated several-fold after recognition of poly I:C as well as invading viruses. This enhanced expression/secretion confers an antiviral state within the cells, leading to the production of Mx protein, as well as in the environment of the stimulated cells (Katze et al., 2002; Jensen and Thomsen, 2011). Poly I:C also modulates the adaptive immune response of animals (Mian et al., 2013). Through its IFN-inducing capabilities, it matures and activates dendritic cells (DCs) and macrophages (MΦ) into competent antigen presenting cells (APC) (McCartney et al., 2009; Mian et al., 2013). Indeed, poly I:C was used in several studies as the sole adjuvant to boost the adaptive immune response (Mian et al., 2013).

Potentially due to their important role in nucleic acid recognition, RLRs family members such as RIG-I/MDA5 are highly conserved during vertebrate evolution and play a key role in the antiviral response against RNA viruses. The loss of function of any one member may impair the innate antiviral response (Xu et al., 2016). Interestingly, RIG-I and MDA5 showed differential recognition capabilities of RNA and DNA viruses. RIG-I primarily recognizes single-stranded negative-strand RNA viruses and some positive-strand RNA viruses, whereas MDA5 seems to recognize mainly positive-strand RNA viruses (Feng et al., 2012; Sato et al., 2015). RIG-I stimulates cytokine production in response to different virus families, including Flaviviridae(Fredericksen et al., 2008; Kato et al., 2008; Loo et al., 2008; Carneiro et al., 2017), Paramyxoviridae (Kato et al., 2005; Yoneyama et al., 2005; Habjan et al., 2008; Loo et al., 2008), Orthomyxoviridae (Kato, Takeuchi, Mikamo-Satoh, et al., 2008; Loo et al., 2008), Rhabdoviridae (Kato et al., 2005; Yoneyama et al., 2005), and Arenaviridae (Habjan et al., 2008). In contrast, MDA5 has been described to be crucial in the recognition of picornaviridae, such as Foot-and-mouth disease virus (FMDV) and Mengo virus (Gitlin et al., 2006; Li et al., 2018; Kato et al., 2008). Some single-stranded (ss) RNA viruses are recognized by both, RIG-I and MDA5 and include Dengue virus, West Nile virus, mouse Hepatitis virus, and Paramyxoviruses., leading to the activation of both PRRs (Fredericksen et al., 2008; Loo et al., 2008; Kato et al., 2011). In addition to the recognition of ssRNA, both of these RLRs are also involved in recognition of double-stranded (ds)RNA (Kato, Takeuchi, Mikamo-Satoh, et al., 2008). Though RIG-1 & MDA5 shared structural homology and signalling pathway, ligand structure and composition also influence their recognition (Loo et al., 2008). RIG-I preferably recognize short, double stranded, 5′ppp bearing pan-handle shaped RNA molecules of 21–27 bp length, while long dsRNA molecules (> 1 kb) selectively stimulate MDA5 molecules (Feng et al., 2012). Moreover high concentration of short dsRNA (∼100bp) molecules also stimulate MDA5 significantly (Peisley et al., 2011; Feng et al., 2012).

Cattle and buffalo are two important dairy animals in India. Buffalo contribute more than 50 % to Indian milk production. In addition, due to their inherent genetic resistance to some common infectious diseases, farmers prefer rearing Buffalos in some geographical areas (Biswal et al., 2012; Yang et al., 2012). The assumption that the immune responses in cattle and buffalo are similar has led to the use of many vaccines and antibiotics, originally developed for use in cattle, efficiently in Buffalos as well (Grandoni et al., 2017). However, there are also clear differences in the response of both ruminants to infectious pathogens (Yang et al., 2012), which require Buffalo-specific research and treatment strategies to be developed. Here, stimulation of innate immune receptors through stimulation of anti-viral responses presents a strategy for the treatment of emerging diseases in ruminants. Thus, the present study was conducted to evaluate potential differential responses in three different cell types (Fibroblast, Monocyte and Macrophage) of water Buffalo (Bubalus bubalis).