PLoS One. 2016 Oct 6;11(10):e0163890. doi: 10.1371/journal.pone.0163890.

Transcriptome Profile of the Chicken Thrombocyte: New Implications as an Advanced Immune Effector Cell.

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Thrombocyte: More than Immune Effector Cell

Platelets, both enucleated in mammals and nucleated in lower vertebrates (thrombocytes), are the source of crucial mediators in hemostatic functions. These cells have been known to be key players in blood clotting and wound healing for many decades. In the last 15-20 years, platelets have been increasingly discussed as cells that have roles beyond hemostasis and thrombosis. These cells contain several biologically active molecules such as cytokines, chemokines, and other immunoregulatory receptors and molecules (1, 2). With the presence of pathogen recognition receptors such as TLRs and the ability to release/produce pro-inflammatory molecules, platelets have the ability to modulate at least innate immune responses (1, 2). There has been much more research done with mammalian platelets, so the role of these cells in physiological function, and capability to be involved in the immune response, is better understood compared to thrombocytes. Enucleated platelets and their precursor cells have been shown to have the ability to regulate adaptive humoral immune responses via their expression and secretion of CD40/CD40L molecules (3). However, the role of thrombocytes in the adaptive side of the immune response has not been studied enough and is not very clear.

 

Our lab in the past 10 years has done significant work to establish thrombocytes (nucleated platelet) as innate responders (1, 4-9). The PLOS ONE publication featured here was the first-ever paper to report a complete thrombocyte transcriptome by examining RNAseq data obtained from thrombocytes isolated from chickens that were control (not treated) and treated with LPS for 1 hour (9). These data revealed which biological processes/biochemical pathways these cells are capable of utilizing as effector immune cells. With further examination of the data beyond the scope of that manuscript, we are able to demonstrate that these thrombocytes are capable of functioning as more than innate immune cells.

 

Thrombocytes, similar to the enucleated counterpart (platelets), possibly are involved in the adaptive immune response or bridging innate and adaptive immune responses. Unlike platelets, that only express major histocompatibility complex (MHC) I (2), further analysis of chicken thrombocyte RNAseq data (8, 9) demonstrates presence of transcripts of molecules associated with MHC I and II (Figure 1). MHC I is generally found on most nucleated cells, while MHC II is exclusively limited to true antigen presenting cells (APC). Endogenous antigens (partially degraded intracellular proteins) processed into peptides by the proteasome within the cytosol are assembled through MHC I to be presented to CD8+ T-cytotoxic (TC) cells. Here, we demonstrate that thrombocytes express transcripts for many genes associated with the endogenous antigen processing pathway such as BF2, B2M, transporter associated with antigen processing (TAP) molecules (TAP1/2, TAPBP), molecular chaperon calnexin (CANX), and others (Figure 1). In addition, these transcripts (BF2, B2M, CANX, HSPA5, TAP1/2, and TAPBP) are represented in red, burnt orange or brown in Figure 1, which indicate comparatively high abundance. Thrombocytes also express transcripts associated with the exogenous antigen processing pathway that specialized antigen presenting cells use to present peptides derived from proteins that the cell has endocytosed. Endocytosed or phagocytosed antigens are degraded into peptides within compartments of the endocytic pathway. The processed peptide then exchanges place with CLIP associated with MHC II in the endocytic pathway, which is then transported to the plasma membrane for presentation to CD4+ T-helper (TH) cells. Here, we were able to detect MHC class II beta chain (BLB1), DM alpha chain (DMA), the cell surface form of the invariant gamma chain (CD74), cathepsin B (CTSB), and others (Figure 1). Again, transcripts for these important molecules are in relatively higher abundance compared to some of the other transcripts shown in Figure 1. Overall, there is relatively more of the transcripts that are higher in abundance (red, burnt orange, brown) than yellow and green, which represent lower abundance (Figure 1). This finding supports the idea that there is constitutive expression of these transcripts associated with MHC I and II processing.

 

In order to verify the presence of the MHC II transcript, we examined for presence of surface expression of MHC II on chicken thrombocytes (Figure 2). In addition to MHC II, surface expression of co-stimulatory molecules CD40, 80 and 86 were also detected on unstimulated chicken thrombocytes. Presence of these markers on unstimulated thrombocytes also indicated constitutive expression. CD40 is generally found on dendritic cells and most B-cell lineages, while constitutive expression of CD80 and CD86 is seen on the professional APC dendritic cells. Therefore, constitutive expression of MHC II and these co-stimulatory molecules indicates that thrombocytes are unconventional/unique immune cells that not only resemble innate effector cells in function but also may have a role in affecting adaptive immunity through cellular contact and interaction with lymphocytes.

 

Expression of transcripts for CD40, CD80 and CD86 varied (up/down-regulated or remained unchanged) when these cells were exposed to different bacterial or viral pathogenic compounds (8). In our Journal of Immunology paper (8), one-hour exposure to lipotechoeic acid (LTA) and lipopolysaccharide (LPS), which are agonist for TLR 2 and 4, respectively, up-regulated (fold change ≥0.5) expressions of CD40, CD80 and CD86. However, one-hour exposure to synthetic analogs of viral RNA {polyinosinic-polycytidylic acid [Poly(I:C)] and thymidine homopolymer phosphorothioate oligonucleotide [Poly(dT)], which are respective agonists for TLR 3 and 7} did not change (fold-change >-0.5 to <0.5) expression of CD40, and CD80. Expression of CD86 remained fairly unchanged with Poly(I:C) exposure while it was up-regulated for Poly(dT). Contrastingly, molecules shown associated with MHC II in thrombocytes in Figure 1 remained unchanged regardless of exposure to any of these four TLR agonists.

 

There has been a report of thrombocytes expressing CD40L among other biologically active surface molecules and receptors (10). Similar to mammalian platelets, the discovery of functional CD40L, especially in light of our recent discovery is of vital importance for the potential modulatory capacity of thrombocytes in bridging innate immunity to the adaptive side of immune responsiveness. Chicken thrombocytes should not have only the potential to be involved in antigen presentation but also the ability to interact with other APC. Besides expressing functional CD40L, platelets and thrombocytes can also release cytokines such as IL-12 during activation by LPS (5, 9). Polarizing cytokines are expressed by APCs that drive naïve CD4+ T-cells to differentiate into TH1 and TH2 cells. Therefore, in addition to attracting APCs, thrombocytes may be able to function like APCs.

The information generated with the thrombocyte model could be of enormous significance for both veterinary and human medical applications related to platelet response at the onset of bacterial and viral infections. In addition, this model system could have translational utility to find orthologous genes related to its enucleated counterpart, the platelet. This information can be particularly useful in understanding the role of platelets in involvement/response to various pathogens associated with different infectious diseases. 

 

 

 

Figure 1:Transcripts of endogenous and exogenous antigen processing pathways found in chicken thrombocytes. This pathway was constructed by analyzing the data from the chicken thrombocyte RNAseq (8, 9) beyond the scope of those manuscripts and by using the PathVisio software (11) and the data. The color code represents abundance of transcripts (average log2 count) where red is highest (>13), burnt orange is high (>11 to 13), brown is medium (>8 to 11), yellow is low (>5 to 8) and green is lowest (<5). Gray molecules are molecules associate with MHC I and II antigen processing pathways that were non-occurring in our dataset.

 

 

 

Figure 2. Presence of MHC II and co-stimulatory molecules on thrombocytes. Chicken thrombocytes were labeled with several monoclonal antibodies. Microscopy image of thrombocytes stained with Alexa Fluor® 594 labeled thrombocyte specific marker, CD41/61(red) counterstained with nuclear stain, 4′,6-diamidino-2-phenylindole (DAPI) (A). Flow cytometric analysis showing surface expression of CD41/61 (B), MHC II (C), CD40 (D), CD80 (E) and CD86 (F). The red curve on the histograms represent cells labeled with the specific primary antibodies while the blue curve represents cells labeled with only Alexa Fluor® 488 (negative control) (B-F). For fluorescent microscopy, thrombocytes were visualized using the Nikon Ti microscope with 60x APO water emulsion objective (Clemson Light Imaging Facility, Clemson University, Clemson, SC). Flow cytometric analysis was performed at the CTEGD Flow Cytometry Core Facility, University of Georgia (UGA), Athens, GA.

 

References:

1. Ferdous, F. and T. R. Scott. 2015. A comparative examination of thrombocyte/platelet immunity. Immunol Lett 163: 32-39.
2. Semple, J. W., J. E. Italiano Jr, and J. Freedman. 2011. Platelets and the immune continuum. Nat Rev Immunol 11: 264-274.
3. Semple, J. W., A. Zufferey, E. R. Speck, K. R. Machlus, R. Aslam, L. Guo, M. J. McVey, M. Kim, R. Kapur, E. 
Boilard, and J. E. Italiano Jr. 2017. OC 58.3. Mature Murine Megakaryocytes Process and Present Both Exogenous and Endogenous Antigens to CD8+ T Cells and Transfer this Ability to Platelets. Research and Practice in Thrombosis and Haemostasis 1: 1-1451.
4. Scott, T. and M. D. Owens. 2008. Thrombocytes respond to lipopolysaccharide through Toll-like receptor-4, and MAP kinase and NF-kappaB pathways leading to expression of interleukin-6 and cyclooxygenase-2 with production of prostaglandin E2. Mol Immunol 45: 1001-1008.
5. Ferdous, F., D. Maurice, and T. Scott. 2008. Broiler chick thrombocyte response to lipopolysaccharide. Poult Sci 87: 61-63.
6. Ferdous, F. and T. Scott. 2015. Bacterial and viral induction of chicken thrombocyte inflammatory responses. Dev Comp Immunol 49: 225-230.
7. Winkler, C., F. Ferdous, M. Dimmick, and T. Scott. 2017. Lipopolysaccharide induced Interleukin-6 production is mediated through activation of ERK 1/2, p38 MAPK, MEK, and NFκB in chicken thrombocytes. Dev Comp Immunol 73: 124-130.
8. Ferdous, F., C. Saski, W. Bridges, M. Burns, H. Dunn, K. Elliott, and T. R. Scott. 2017. Bacterial and Viral Products Affect Differential Pattern Recognition Receptor Activation of Chicken Thrombocytes Evidenced through RNA Sequencing. J. Immunol. 199: 774-781.
9. Ferdous, F., C. Saski, W. Bridges, M. Burns, H. Dunn, K. Elliott, and T. R. Scott. 2016. Transcriptome Profile of the Chicken Thrombocyte: New Implications as an Advanced Immune Effector Cell. PloS One 11: e0163890.
10. Tregaskes, C. A., H. L. Glansbeek, A. C. Gill, L. G. Hunt, J. Burnside, and J. R. Young. 2005. Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate. Dev Comp Immunol 29: 361-374.
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