Chronic enteropathy in dogs: the role of macrophages and preliminary results in inflammatory cytokines

Abstract

Chronic enteropathy (CE) is an umbrella term used in dogs to describe a group of diseases with different aetiologies, characterised by chronic gastrointestinal signs. These diseases are clinically classified according to treatment response as food-responsive (FRE), antibiotic-responsive (ARE), and immunosuppressant-responsive enteropathies (IRE).

The first part of this PhD thesis prospectively describes the features of CE that are commonly seen in dogs presenting to a referral centre in Australia; information that has not been available previously. We found that similar to other countries, most dogs with CE are food-responsive, followed by antibiotic-responsive with a minority of immunosuppressant-responsive. Furthermore, our study raised concerns about prolonged antibiotic treatment for dogs with ARE. Firstly, most of these dogs do not respond to treatment completely for prolonged periods (as opposed to dogs with FRE that do), raising the question about the real benefit of antibiotic treatment. Secondly, half of the dogs with ARE required long-term or pulse treatment with antibiotics, which raises concerns about development of bacterial resistance. Our findings highlight the need to find alternative treatment for dogs with ARE in view of the poor long-term outcome. Although most dogs with FRE had long-term remission, adequate dietary trials were not performed until reaching the referral setting. This indicates that better education of general veterinary practitioners about the importance of performing adequate diet trial is needed to improve early disease remission in these dogs.

The next focus of the research was to evaluate the role of macrophages in CE; this was achieved by using two macrophage markers: calprotectin and cluster of differentiation 163 (CD163) in immunohistochemical examination. Both immunohistochemical markers highlighted two different populations of macrophages in our intestinal biopsy specimens. Overall the number of CD163 positive cells was higher than calprotectin positive cells both in crypts and villi. Dogs with FRE and IRE had a decreased CD163:calprotectin ratio compared to healthy dogs with an increase in the ratio after treatment. Our results suggest that there is an imbalance in macrophage populations in dogs with FRE and IRE, with partial resolution following clinical response characterised by an increase in the ratio CD163:calprotectin. Interestingly, dogs with ARE not only have a poor long-term response, but also have different macrophage populations from dogs with FRE and IRE; and in fact, are very similar to healthy dogs without change in their macrophage populations with treatment response. These results suggest that macrophages play a role in the pathogenesis of FRE and IRE dogs with normalisation of macrophage populations with treatment response.

The CD163 receptor is cleaved during macrophage activation and is released into the circulation as a soluble form. In view of the decreased number of CD163 cells in the intestine of dogs with FRE and IRE at diagnosis (and subsequent increase with clinical remission), we wanted to determine if soluble CD163 could be detected in dog serum, and therefore potentially serve as a biomarker. Two different ELISAs were tested and although one of them showed some signal, further testing of the antibodies used in the assay did not support that the signal was specific for CD163. With this experiment, we were not able to quantify soluble CD163 in dogs, but this molecule retains potential as a biomarker for diagnostic and monitoring purposes in CE as well as in other diseases characterised by macrophage activation.

Biomarkers of systemic inflammation were also assessed in the same cohort of dogs and we showed that serum IL-6 decreased in dogs with CE after resolution of clinical signs. Similarly to soluble CD163, cytokines might play a role in further differentiating between the different causes of CE for prognostic and therapeutic purposes. Future studies are needed to assess these cytokines in a larger cohort of dogs to be able to study differences between FRE, ARE, and IRE, and further assess their role as biomarkers.

Finally, we studied cytokine production by lymphocytes or monocytes in the peripheral blood of healthy dogs, and the effect of different immunosuppressive treatments on cytokine production. Differential activation of lymphocytes or monocytes can easily be achieved by using specific activators in whole blood. The advantage of this technique is that there is minimal handling of the cells with less risk of iatrogenic activation. Cytokine production was affected by cyclosporine and prednisolone, but not by mycophenolate, leflunomide, or azathioprine. Cyclosporine inhibited production of tumour necrosis factor (TNF), interferon gamma and IL-10 by lymphocytes whereas prednisolone inhibited TNF production by both lymphocytes and monocytes. Our findings suggest that this methodology can be used to monitor dogs treated with both drugs concurrently – although this needs to be further assessed with future studies.

Future studies highlighted by our research suggest more in-depth assessment of serum cytokines as biomarkers for dogs with CE not only for monitoring purposes, but to determine if different patterns of cytokines can be useful to refine the classification of CE. Similarly, whole blood stimulation can be used to better assess underlying priming of the immune system and to monitor treatment response. Finally, our findings suggest that macrophages play a significant role in the pathophysiology of CE in dogs, particularly in FRE and IRE, but additional work is required to better understand their function in CE.