Pathogen associated molecular pattern-induced TNF, IL-1β, IL-6 and CXCL-8 production from feline whole blood culture

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Abstract

Whole blood culture (Cwb) is a method to evaluate leukocyte response to stimuli. We used Cwb to evaluate the inflammatory response to pathogen associated molecular patterns (PAMPs) in cats. Blood was collected from diluted with RPMI and stimulated with various concentrations of lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PG) or control (PBS). Multiple concentrations of LPS, LTA and PG significantly stimulated tumor necrosis factor (TNF), interleukin (IL)-1β and CXC chemokine ligand (CXCL)-8 in feline Cwb. All PAMPs failed to stimulate IL-6 production and PG failed to stimulate CXCL-8 production. Lipopolysaccharide was a more potent inducer of IL-1β and CXCL-8 than LTA or PG and LTA is a more potent inducer of CXCL-8 than PG. Based on these data, PAMPs from gram positive and negative bacteria induce TNF, IL-1β and CXCL-8 production in feline whole blood. Cats appear to be relatively more sensitive to gram negative compared to gram positive bacteria.

Introduction

Sepsis, the systemic inflammatory response to infection, is a serious problem in cats with mortality rates ranging from 22% to 79% (Waddell et al., 2002, Costello et al., 2004, Sergeeff et al., 2004, Barrs et al., 2005). Sepsis has been associated with many conditions including septic peritonitis, hepatic abscessation, pyothorax, bacteremia, pneumonia, endocarditis, pyelonephritis and pyometra in the cat (Brady et al., 2000, Waddell et al., 2002, Costello et al., 2004, Sergeeff et al., 2004, Barrs et al., 2005). Although cats develop many of the classic clinical findings associated with sepsis, they also develop relatively unique manifestations including bradycardia, hypothermia and abdominal pain (Brady et al., 2000, Waddell et al., 2002, Sergeeff et al., 2004). Information about pathophysiology of sepsis in cats, especially the pathophysiology of these unique manifestations, is lacking. Current therapy consists of infection control and aggressive supportive care but cats may also benefit from therapies aimed at blunting the inflammatory response during sepsis. One of the barriers to developing novel anti-inflammatory strategies for sepsis in cats is the limited understanding of the pathophysiology of sepsis in this species.

Whole blood culture (Cwb) is an inexpensive and rapid ex vivo method for studying leukocyte responsiveness to stimuli and has been used to evaluate the inflammatory response and novel anti-inflammatory therapies in other species (Figueiredo et al., 2008, Al-Hanbali et al., 2009, Damsgaard et al., 2009, Wu et al., 2009). Whole blood culture maintains interactions between populations of blood cells and plasma, which more appropriately mimics physiological conditions in vivo, as compared with isolated peripheral blood mononuclear cells (PBMCs). Isolated PBMCs contain a mixture of T cells, B cells, NK cells and monocytes and have been shown to have a distorted monocyte to lymphocyte ratio in an isolated sample as compared to whole blood (De Groote et al., 1992, Damsgaard et al., 2009). Isolated cells also lack cytokines, growth factors and other cell types that would be normally present in whole blood (Albers et al., 2005, Damsgaard et al., 2009). Plasma factors are particularly important since they are necessary for lipopolysaccharide (LPS) and peptidoglycan (PG) to activate leukocytes in some species (Mattsson et al., 1994, Benbarek et al., 1998). Moreover, Cwb is a less technically demanding technique and requires smaller volumes of blood than methods involving isolated PBMCs.

Whole blood culture has been used to evaluate LPS-induced tumor necrosis factor (TNF) production from feline whole blood previously (Otto and Rawlings, 1995, Aguirre et al., 2003). However, other pathogen associated molecular patterns (PAMPs) like lipoteichoic acid (LTA) from gram positive bacteria and PG primarily from gram positive bacteria, but also from gram negative bacteria, and production of inflammatory mediators other than TNF have not been evaluated. Since the type of organism (i.e. gram positive vs. gram negative bacteria) influences the inflammatory response in other species understanding the unique features of the inflammatory response to various PAMPs in cats would be valuable (Cui et al., 2000). The purpose of this study was to evaluate the inflammatory response to LPS, LTA and PG in cats using a feline whole blood culture system.

Section snippets

Animals

Six adult, purpose bred cats (Liberty Research, Waverly, NY) were used. Animals were cared for according to the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals and the study was approved by the Animal Care and Use Committee at the University of Missouri. The cats were maintained on commercial adult cat food and water ad libitum. The cats had been instrumented with subcutaneous implantable vascular access ports (Norfolk Vet Products, Skokie, IL) one year prior to

TNF activity in whole blood culture

Tumor necrosis factor production was significantly greater from whole blood stimulated with LPS, LTA and PG (Fig. 1) compared to control. All concentrations of LPS (p < 0.007), LTA (p < 0.002) and PG (p < 0.02) tested resulted in significant TNF production from feline whole blood. The degree of supernatant TNF activity was not stimulant concentration dependent for LPS and LTA. For PG, 1 ng/ml stimulated significantly less TNF activity than the other concentrations (p = 0.001). There was no difference in

Discussion

To our knowledge, this is the first report of LTA or PG-induced inflammatory mediator production in cats and the first dose response evaluation for LPS using whole blood culture. The minimum concentration tested at which LPS or LTA stimulated significant production of TNF, IL-1β and CXCL-8 concurrently was 100 or 1000 ng/ml, respectively. At 1000 ng/ml PG significantly stimulated both TNF and IL-1β production concurrently; however PG simulation failed to result in significant CXCL-8 production,

Conclusion

Feline whole blood culture can be used to investigate PAMP-induced TNF, IL-1β and CXCL-8 production in cats. However, whole blood culture does not appear to be an efficient method to evaluate PAMP-induced IL-6 production or PG-induced CXCL-8 production at the concentrations tested. This whole blood culture system can be used for future studies evaluating the inflammatory response to infection and novel anti-inflammatory therapies in cats, ex vivo.

Pathogen associated molecular patterns from both

Conflict of interest

The authors have no conflicts of interest.

Acknowledgements

The authors would like to thank Norfolk Vet Products for their generous donation of the vascular access ports and Huber needles used in this study and Dr. Leona Rubin for her kind donation of L929 cells. The authors also acknowledge Dr. David Dismukes for his surgical expertise and Dr. Chee-Hoon Chang for her technical assistance with this study. Funding for this study was provided by a University of Missouri Richard Wallace Research Incentive Grant.

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