Comparative Genomic Analysis of Buffalo (<i>Bubalus bubalis</i>) NOD1 and NOD2 Receptors and Their Functional Role in <i>In-Vitro</i> Cellular Immune Response

<div><p>Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated <i>in-vitro</i> cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated <i>in-vitro</i> immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.</p></div

Inductive mRNA expression of NOD1, NOD2, interleukins and IFN-γ in iE-DAP and MDP treated alveolar macrophages of buffalo.

<p>X-axis represents the time intervals following agonists addition, Y axis shows relative fold change in mRNA expression of genes over respective controls (sh-controls). Columns indicated with asterisks (*) differ significantly (p<0.05) from their respective controls. Protein expression of NOD1, NOD2 and β-actin in treated cells over different time intervals has been shown also.</p

Inductive mRNA expression of NOD1, NOD2, RICK, NF-κB and IL-8 in iE-DAP and MDP treated mammary epithelial cell line of buffalo.

<p>X-axis represents the time intervals following agonist addition, Y axis shows relative fold change in mRNA expression of genes over respective controls (sh-controls). Columns indicated with asterisks (*) differ significantly (p<0.05) from their respective controls. Protein expression of NOD1, NOD2 and β-actin in treated cells over different time intervals has been shown also.</p

Amino acid conservation of different domains of NOD1 and NOD2 among different species.

<p>Estimates of evolutionary divergence among sequences were conducted by calculating pair-wise distances using the JTT matrix-based model. The zone marked with green lines spans comparison of respective region of NOD1 and NOD2 in different species. The blue or violet lines span the zones forinter-species comparison of a region specific to either NOD1 or NOD2. (A) The map indicated NOD2 CARDs are well conserved among different species, NOD1 CARD is less conserved and there were little similarities amongst the different CARDs in the species under consideration. (B) NOD1 NACHT is well conserved among different species compared to NOD2 NACHT. There was distant correlation amongst the NACHT domains of NOD1 and NOD2 in species under consideration. (C) Distances among LRRs of NOD1 and NOD2 of different species. Each small triangle (example marked with black lines) or square represent nine species under study (for clarity of the figure, species names have not shown). (D-F) Cartoon representations showing conserved amino acids responsible for forming basic (blue) and acidic (red) patches on three cards. Residues D42, D48, E53, D54, and E56 have been implicated for CARD-CARD interaction of NOD1 and RICK. Ubiquitinylation sites E85 (pink) was conserved Y89 was substituted with histidine that was found to be was buried by side chains of L93, Y96 and L100. Residues R38 and R86 required for NOD2 CARD-I and RICK CARD interaction were also conserved in buffalo. Residues implicated for ubiquitinylation in NOD2 CARDs have been also shown with their electron density spheres.</p

Constitutive expression and localization pattern of buffalo NOD proteins.

<p>Constitutive protein expression of buffalo NOD1 and NOD2 in different tissues detected by Western blot using equal amount of proteins from the tissues.</p

Comparison of LRR domains of buNOD1 (left panel) and buNOD2 (right panel).

<p>Ten putative LRRs were identified in both NOD1 and NOD2 sequences of buffalo. Conserved amino acids responsible for ligand binding and interaction were mapped to models and highlighted in the alignment. Residues indicated in models but not shown in alignment were located in the intermediate loop region of turn and helix. Residues H788, K790, G792, E816, G818, W820, W874 of NOD1 LRRs, responsible for iE-DAP binding, formed a cluster at central concave face. Residues important for NOD2’s response to MDP (highlighted green and red spheres) were located in β-sheet/ β-sheet turns, loop regions as well as in the α-helices, while residues involved in MDP binding (red) were confined to the β-strand/ β-turn motif at the central concave face of LRR.</p

Inductive mRNA expression of NOD1, NOD2, downstream adapter (RICK), and effector (interleukins and interferons) in iE-DAP and MDP treated PBMCs.

<p>X-axis represents the time intervals following agonists addition, Y axis shows relative fold change in mRNA expression of genes over respective controls (sh-controls). Columns indicated with asterisks (*) differ significantly (p<0.05) from their respective controls. Protein expression of NOD1, NOD2 and β-actin in treated cells over different time intervals has been shown also.</p

Inductive mRNA expression ofNOD1, NOD2, RICK, NF-κB and IL-8 in iE-DAP and MDP treated foetal fibroblast cells of buffalo.

<p>X-axis represents the time intervals following agonist addition, Y axis shows relative fold change in mRNA expression of genes over respective controls (sh-controls). Columns indicated with asterisks (*) differ significantly (p<0.05) from their respective controls. Protein expression of NOD1, NOD2 and β-actin in treated cells over different time intervals has been shown also.</p