Sib Sankar Giri^a, c, Shib Sankar Sen^b, Jin Woo Jun^a, Se Chang Park^a,* V. Sukumaran^{c, d, *}

^a Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151742, South Korea

^b School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

^c Dept. of Biotechnology, Periyar Maniammai University, Thanjavur 613403, Tamil Nadu, India

^d Dept. of Zoology, Kundavai Nachiyar Government Arts College for Women (Autonomous), Thanjavur 613007, Tamil Nadu, India

^*Corresponding authors :  Se Chang Park, V. Sukumaran

Abstract

Present study was undertaken to investigate the efficiency of heat-killed whole-cell products (HKWCPs) of probiotic Pseudomonas aeruginosa VSG2 strain in stimulating the cytokine responses in the head kidney (HK) macrophages of Labeo rohita. The HK macrophages were incubated with HKWCPs or lipopolysaccharide (LPS), and the responses of cytokine genes, namely interleukin-10 (IL-10), IL-1β, IL-p35, IL-12p40, tumour necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), interferon-alpha (IFN-α), and interferon-gamma (IFN-γ) were assessed by quantitative real-time PCR (qRT-PCR) at 2, 8, 16, 24, 48, 72 hours post-stimulation (hps). Among proinflammatory cytokines, significantly higher ____expression____ of IL-1β and TNF-α was observed at 8 - 24 hps, and 2 - 16 hps with HKWCPs, respectively, as compared to controls. However, COX-2 and NF-κB displayed strong ____expression____ (P < 0.05) at 2 - 8 hps, and 8, 16 and 72 hps with HKWCPs, respectively. Antiviral cytokines IFN-α and IFN-γ displayed strong ____expression____ (P < 0.05) at 8 - 24 hps, and 2, 24 and 48 hps with HKWCPs, respectively. ____Expression____s of cell-mediated immune factor genes (IL-12p35 and IL-12p40) were also significantly upregulated at various time points, except IL-12p40 at 72 hps, in HK macrophages stimulated with HKWCPs. ____Expression____ of the anti-inflammatory cytokine IL-10 was upregulated (P < 0.05) at 2 - 24 hps HKWCPs, compared to controls. Enhanced cellular (phagocytic activity and superoxide anion production) and humoral (lysozyme) immune parameters of treated HK macrophages confirmed the induction of inflammatory response. Thus, our results indicated that HKWCPs of probiotic P. aeruginosa VSG2 had greater potential for stimulating the in vitro ____expression____ of cytokines in fish and that these HKWCPs may be used as vaccine adjuvants in aquaculture.

Keywords : HKWCPs; Pseudomonas aeruginosa VSG2; LPS; cytokine response; macrophage; Labeo rohita