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Chemotherapy and remission status do not alter pre-existing innate immune dysfunction in dogs with lymphoma
Research in Veterinary Science 97 (2014) 230–237
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Chemotherapy and remission status do not alter pre-existing innate immune dysfunction in dogs with lymphoma S. Axiak-Bechtel a, B. Fowler b, D.H. Yu c, J. Amorim b, K. Tsuruta b, A. DeClue b,* a Comparative Oncology and Epigenetics Laboratory, Department of Veterinary Medicine and Surgery, University of Missouri, 900 East Campus Drive, Columbia, MO 65211, USA b Comparative Internal Medicine Laboratory, Department of Veterinary Medicine and Surgery, University of Missouri, 900 East Campus Drive, Columbia, MO 65211, USA c College of Veterinary Medicine, Chonnam National University, 77 Yongbong-ro, Buk-gu, 17 Gwangju, Korea
A R T I C L E
I N F O
Article history: Received 12 February 2014 Accepted 20 July 2014 Keywords: Innate immunity Lymphoma Chemotherapy Neutrophils
A B S T R A C T
Dogs with lymphoma have altered innate immunity and little is known about the effects of chemotherapy on innate immune function in dogs. Lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PG) – induced leukocyte cytokine production capacity, and phagocytosis and respiratory burst were evaluated in dogs prior to and following 6 weeks of chemotherapy. Dogs had decreased TNF production following LPS stimulation and increased IL-10 production following PG stimulation, which did not improve following remission of lymphoma. Dogs also had reduced E. coli-induced respiratory burst function after chemotherapy induced complete or partial remission. Dogs with lymphoma have an imbalance in proand anti-inflammatory cytokine production which did not improve with remission, and, following treatment, a decrease in respiratory burst function. Altered immune responses following exposure to bacterial pathogen associated molecular pattern motifs and bacteria may have many implications in the management of canine lymphoma. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lymphoma is the most common hematopoietic tumours in dogs (Dorn et al., 1970; Vail and Young, 2013). Despite the high initial response rate to chemotherapy, almost all affected dogs eventually relapse and ultimately die of lymphoma (Garrett et al., 2002; Vail and Young, 2013). The most common dose limiting adverse effect of chemotherapy is myelosuppression and subsequent immunosuppression, leading to increased risk of infection (Britton et al., 2012; Garrett et al., 2002; Vail and Young, 2013; Vaughan et al., 2007). Since quality of life is an important factor in determining treatment protocols for dogs, concerns with increased risk of infection have led to reductions of chemotherapy dose intensity. As a result, dogs receive lower than maximally tolerated doses of chemotherapy (Vaughan et al., 2007). Compounding these factors, dogs with lymphoma have an increased risk of developing sepsis with chemotherapy compared with dogs with other tumour types (Sorenmo et al., 2010). In humans, a chemotherapy dose reduction of 10% results in significantly reduced survival times (Pettengell et al., 2008). Similarly, dogs receiving maximum tolerated doses of chemotherapy as
* Corresponding author. Tel.: +1 573 882-7821; fax: +1 573 884-7563. E-mail address: [email protected] (A. DeClue). http://dx.doi.org/10.1016/j.rvsc.2014.07.009 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
indicated by significant myelosuppression (neutropenia) have a longer remission duration compared with dogs that do not experience myelosuppression (Vaughan et al., 2007). This implies that selective dose intensification may be associated with better outcome. However, increasing chemotherapy dose intensity in dogs may also lead to increased risk of sepsis and a reduction in quality of life, with chemotherapy induced sepsis having an 8.5% mortality rate (Britton et al., 2012). In addition to decreasing the number of phagocytic cells available to fight infection, induction chemotherapy will decrease the percentage of neutrophils capable of performing oxidative burst compared with pre-treatment (Leblanc et al., 2013); dogs with lymphoma also have an impaired ability to produce inflammatory cytokines in response to pathogen associated molecular patterns (PAMPs) (Fowler et al., 2011). These studies indicate that greater understanding of preexisting and chemotherapy-induced immunodysfunction is needed in dogs with lymphoma prior to pursuing dose intensification. Our objective was to investigate the effect of chemotherapy on leukocyte cytokine production capacity and circulating polymorphonuclear cell (PMN) phagocytosis and respiratory burst function. We hypothesized that dogs with lymphoma would have blunted leukocyte cytokine production capacity, PMN phagocytosis, and PMN respiratory burst compared with control dogs and that these immune function parameters would improve following chemotherapy induced remission.
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2. Materials and methods 2.1. Patient population This project was approved by the University of Missouri Animal Care and Use Committee (protocol #7334). Client owned dogs presenting to the University of Missouri, Veterinary Medical Teaching Hospital with a cytologic or histopathologic diagnosis of lymphoma were eligible for enrolment with informed owner consent. All dogs had a history, physical examination, and complete blood count (CBC) as part of routine diagnostics and staging. Other staging procedures routinely recommended, but not required, for dogs with lymphoma in this study included a biochemical profile, urinalysis, chest radiographs, abdominal ultrasound, bone marrow aspirate, and immunophenotyping. Dogs whose owners elected to use a standard of care, a six-month combination chemotherapy protocol (Garrett et al., 2002), were eligible for enrolment (Table 1). Following chemotherapy treatment, remission status was determined based on physical examination and CBC. The healthy control group consisted of dogs belonging to faculty, staff, and students at the University of Missouri. Dogs were deemed healthy based on history, physical examination, complete blood count, and plasma biochemical analysis. Dogs were staged based on the World Health Organization’s clinical staging system for lymphoma in domestic species. Dogs with a history of immunosuppressive therapy (including glucocorticoids, chemotherapy, or radiation therapy) in the month prior to presentation were excluded from the study. Additional exclusion criteria were previously diagnosed immune system disorder or history of inflammatory disease within three months of presentation. Dogs in the lymphoma group that failed to complete the first five treatments of chemotherapy were also excluded. Blood was collected into potassium EDTA, lithium heparin, and sodium heparin tubes for a CBC, biochemical profile, and immunologic evaluation, respectively. Dogs with lymphoma had blood collected at presentation prior to treatment (baseline) and again at week 6 of chemotherapy, prior to chemotherapy administration. Week 6 was defined as the week dogs were scheduled to begin the second cycle of treatment with vincristine. Therefore, this was at least 2 weeks after the first doxorubicin treatment and at least 1 week after discontinuation of prednisone. Remission status was recorded and based on physical examination findings and complete blood counts. Collection from control dogs occurred at baseline and again 6 weeks later to determine if time related fluctuations in immune parameters occurred. Samples for CBC, plasma biochemical profile and phagocytic function were processed within 12 hours of collection, and samples for cytokine evaluations processed within 2 h of collection. 2.2. Leukocyte cytokine production capacity Leukocyte cytokine production was performed as previously described (DeClue et al., 2008; Deitschel et al., 2010; Fowler et al., 2011). Five millilitres of whole blood was diluted 1:2 with complete Roswell Park Memorial Institute medium containing 200 U/ml of
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penicillin, 200 mg/ml of streptomycin (Gibco®, Invitrogen, Grand Island, NY, USA), placed in 12-well plates, and stimulated with the following pathogen associated molecular pattern motifs: lipopolysaccharide (LPS) from Escherichia coli (E. coli) O127:B8 (final well concentration, 100 ng/ml; Sigma-Aldrich, St. Louis, MO, USA), lipotechoic acid (LTA) from Streptococcus faecalis (final well concentration, 1000 ng/ml; Sigma-Aldrich), peptidoglycan (PG) from Staphylococcus aureus (final well concentration, 1000 ng/ml; SigmaAldrich), or control phosphate buffered saline (PBS). Wells were gently mixed on a plate rocker for 5 min, then incubated for 24 h at 37 °C and 5% CO2. The supernatant was then collected and stored at −80 °C until analysis. Tumour necrosis factor (TNF), interleukin (IL)-6, and IL-10 were measured in the supernatant using a canine specific multiplex bead based assay (Millipore, Billerica, MA, USA) (Karlsson et al., 2012). 2.3. Phagocytosis and respiratory burst function Granulocyte phagocytic and respiratory burst function tests were determined using commercially available test kits (Phagotest® and Phagoburst®, Orpegen Pharma, Heidelberg, Germany) previously validated for dogs (LeBlanc et al., 2010). For phagocytic function tests, 100 μl of whole blood was incubated with either 20 μl of washing solution (negative control) or opsonized, FITC-labelled E. coli (1 × 109 bacterial per ml) for 10 min, and extracellular fluorescence was removed by adding 100 μl of quenching solution (1×). Samples were washed using 3 ml of washing solution three times followed by incubation with 2 ml of fixing solution for red blood cell lysis and cell fixation. Granulocytes with phagocytized E. coli were measured by flow cytometry after DNA staining using propidium iodide. For the measurement of oxidative burst, 100 μl of whole blood was incubated with 20 μl of washing solution (negative control), opsonized E. coli (1 × 109 bacterial per ml), or phorbol myristate acetate (PMA, 0.0081 mM) for 10 min. Formation of reactive oxidants during oxidative burst was monitored by the addition and oxidation of 20 μl dihydrorhodamine 123 to Rhodamine 123 (1× solution). The reaction was stopped by addition of 2 ml of fixing and RBC lysing solution. Samples were then washed using 3 ml of washing solution. A DNA staining solution (propidium iodide, 1× solution) was then added and oxidative burst capacity was determined using flow cytometry. 2.4. Flow cytometry Flow cytometry was performed at the University of Missouri Cell and Immunology Core Facility using the CyAn ADP machine and summit software. DNA positive cells were identified on an FL2 histogram plot. These cells were then applied to a forward vs side scatter plot. Then granulocytes were identified and applied to an FL1histogram (Fig. 1). Percentage of FITC positive granulocytes having performed phagocytosis was recorded for assessment of phagocytic function. The percentage of Rhodamine 123 positive granulocytes having produced reactive oxygen metabolites was recorded for assessment of respiratory burst function. A minimum of 10,000 total events were recorded for each sample. 2.5. Statistical analysis
Table 1 Chemotherapy protocol used for dogs in this study. Week
0
L-asparaginase (400 IU/kg) Vincristine (0.5–0.7 mg/m2) Cyclophosphamide (200–250 mg/m2) Doxorubicin (1 mg/kg if <15 kg or 30 mg/m2 if >15 kg) Prednisone (tapering, starting at 2 mg/kg/day )
X
1
2
X
3
4
X X
X
X
X
6 X
X
X
5
X
Statistical analysis was performed using commercially available software (SigmaStat, Systat Software Inc.). Data distribution properties were tested using histogram plots. Differences in white blood cell count between the lymphoma and control groups at week 1 and 6 and between weeks in the lymphoma group were compared using two way repeated measures ANOVA and Fisher LSD method. Difference in variables between week 1 and week 6 in the control and lymphoma groups were compared using a Wilcoxon
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Fig. 1. Gating scheme for flow cytometry. First PI positive cells were identified, gated, and applied to a forward vs side scatter plot. Then a gate was drawn around neutrophils on the forward vs side scatter plot and applied to a FITC histogram.
signed rank test. Variables for dogs that achieved complete remission versus partial remission at week 6 in the lymphoma group were compared using a Mann–Whitney rank sum test. Multiple linear regression was used to evaluate the association among white blood cell count, neutrophil count, monocyte count, and cytokine production. A P-value of < 0.05 was considered statistically significant. Outliers were defined as any value greater than 1.5 times the interquartile range above or below the first or third quartile and were excluded from graphical presentation. 3. Results 3.1. Patient population Eleven control dogs were enrolled. Ages ranged from 2 to 10 years and the median age was seven years. Breeds enrolled were Australian Shepherd (n = 3), Labrador retriever (n = 3), Border collie (n = 2), and one each of German Shepherd Dog, Newfoundland, and Golden retriever. Weight ranged from 20 to 45 kg with a median of 30.5 kg. No noteworthy abnormalities were present on CBC or biochemical profile in any dog. Seventeen dogs with lymphoma were enrolled. Four dogs were excluded because they did not complete required treatment (n = 3) or they died prior to week 6 (n = 1). The ages of the remaining 13 dogs ranged from 4 to 14 years old and the median was eight years. Breeds included Beagle (n = 2), Labrador retriever (n = 2), mixed breed (n = 2) and one each of Rottweiler, Cocker Spaniel, Bull Terrier, Australian Shepherd, Golden Retriever, Border collie, and Schnauzer. Weight ranged from 10.8 to 49.2 kg and the median weight was 28.25 kg. Eleven dogs were staged with a CBC, chemistry panel, urinalysis, chest radiographs, abdominal ultrasound, and bone marrow
aspirate. All 11 of these dogs were immunophenotyped as B cell lymphoma using histopathology and immunohistochemistry. These dogs were considered stage IIIa (n = 3), IVa (n = 3), IVb (n = 2), Va (n = 1), and Vb (n = 2). Two of the 13 dogs were staged with CBC, chemistry panel, urinalysis, chest radiographs, and abdominal ultrasound, but no bone marrow aspirate. Both of these dogs were considered stage Va based on circulating lymphoblasts found on CBC. One dog’s immunophenotyping was consistent with B cell lymphoma and the other dog was not immunophenotyped because this diagnostic was declined by the pet owner. At the time of second blood sample collection, all dogs had received, in this order, L-asparaginase, prednisone, vincristine, cyclophosphamide, vincristine, and doxorubicin. The sample was collected at least 2 weeks after doxorubicin and at least 1 week after discontinuation of prednisone. Because some dog owners travelled long distances for chemotherapy appointments, dogs that were neutropaenic on a referring veterinarian CBC had treatment, and therefore their week 6 appointment, delayed by 1 week. Therefore, no dog was neutropaenic at the time of second sample collection. Six of the 13 dogs with lymphoma were in complete remission at the time of second sample collection, and seven dogs were in partial remission based on examination and CBC. The median and ranges of total white blood cell, neutrophil, monocyte, and lymphocyte counts are shown in Table 2. Dogs with lymphoma had a higher total white blood cell count and neutrophil count than control dogs at week 1 and 6. 3.2. Stimulated leukocyte cytokine production capacity In control dogs, no significant difference was present between week 1 and week 6 leukocyte production of: IL-6 when
Table 2 White blood cell, neutrophil, monocyte and lymphocyte counts for normal dogs and dogs with lymphoma, mean and standard deviation. Total white blood cells (cells/μl) Lymphoma week 1 Mean (standard deviation) Control week 1 Mean (standard deviation) Lymphoma week 6 Mean (standard deviation) Control week 6 Mean (standard deviation) a b
12,278 (5,873)
a
7050 (1,463) 11,350 (4,523) 7844 (2,839)
P ≤ 0.01 compared with control group during same week. P < 0.05 comparing same disease group in different weeks.
Neutrophils (cells/μl)
Monocytes (cells/μl)
Lymphocytes (cells/μl)
429 (345)
2224 (3373)
4555 (982)
434 (190)
1940 (928)
8502 (3,474)a
782 (518)a,b
1346 (615)
4846 (1,625)
340 (350)
1432 (723)
9560
(8,683)a
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Fig. 2. Comparison of leukocyte lipopolysaccharide-induced TNF production (A) and peptidoglycan-induced IL-10 production (B) from healthy dogs and dogs with lymphoma at week 1 (baseline) and week 6 of chemotherapy. Data are presented as a box and whisker plots. The upper and lower edges of the box represent the 75th and 25th percentiles respectively; the line within the box is the median value. Whiskers represent the range. a Baseline samples are significantly different between lymphoma and control groups at the same time point.
stimulated with LPS, LTA, or PG; IL-10 when stimulated with LPS, LTA, or PG; or TNF when stimulated with LPS, LTA, or PG. Compared with controls, dogs with lymphoma had blunted production of TNF following stimulation with LPS (p = 0.008; Fig. 2A). Dogs with lymphoma also produced significantly more IL-10 following stimulation with PG compared with control dogs (p = 0.015; Fig. 2B). There were no other differences in LPS, LTA, or PG induced TNF, IL-6 or IL-10 between control dogs and dogs with lymphoma. Lipopolysaccharide, LTA, or PG-induced leukocyte production of TNF, IL-6, or IL-10 was not significantly different between baseline and week 6 in the lymphoma group. Cytokine production at week 6 in dogs that achieved complete remission was not different that dogs that achieved only a partial remission (Table 3). At week 1, dogs with lymphoma had a significantly lower LPS induced TNF to IL-10 ratio (p = 0.022) and PG induced TNF: IL-10 ratio (p = 0.042) than control dogs (Fig. 3). When comparing these ratios between pre- and post-treatment in dogs with lymphoma, dogs treated with chemotherapy had a significantly higher LPS induced TNF: IL-10 ratio (p = 0.014; Fig. 3) when compared with baseline. No difference was found in TNF:IL-10 ratio for LTA
Table 3 Comparison of LPS, LTA and PG stimulated TNF, IL-6, IL-10 production and TNF:IL10 production ratio, phagocytosis of E. coli, E. coli-induced respiratory burst and PMAinduced respiratory burst between dogs with complete and partial remission after 6 weeks of chemotherapy. Data represented as median (Q1; Q3) in pg/ml (cytokines) or % cells (phagocytosis and respiratory burst). There were no differences in cytokine production between dogs in complete and partial remission. Parameter
Complete remission (n = 6)
Partial remission (n = 7)
LPS TNF LPS IL-6 LPS IL-10 LPS TNF:IL-10 LTA TNF LTA IL-6 LTA IL-10 LTA TNF:IL-10 PG TNF PG IL-6 PG IL-10 PG TNF:IL-10 E. coli phagocytosis E. coli respiratory burst PMA respiratory burst
1295 (565; 2303) 534 (258; 724) 1234 (516; 2418) 0.61 (5.7; 4.2) 1198 (785; 5278) 689 (345; 767) 1620 (553; 4484) 0.55 (0.34; 4.6) 791 (590; 2033) 407 (298; 666) 665 (473; 2303) 0.59 (0.4; 4.2) 91.2 (87.6; 94.3) 62.5 (40.6; 94.8) 97.3 (64.2; 99.3)
864 (697; 1295) 561 (320; 806) 979 (298; 5156) 1.1 (0.5; 2.3) 912 (874; 1299) 464 (348; 881) 1529 (500; 8141) 0.85 (0.31; 1.8) 734 (554; 917) 257 (217; 659) 681 (208; 7212) 1.29 (0.26; 1.68) 89.4 (82.6; 92.1) 69.5 (60.4; 88) 92.3 (67.3; 97.5)
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Fig. 3. Comparison of leukocyte lipopolysaccharide-induced TNF:IL-10 production ratios (A) and peptidoglycan-induced TNF:IL-10 production ratios (B) from healthy dogs and dogs with lymphoma at week 1 (baseline) and week 6 of chemotherapy. See Fig. 2 for rest of key. a Baseline samples are significantly different between lymphoma and control groups at the same time point. b Samples are significantly different between week 1 and week 6 within the lymphoma group.
stimulation or between week 1 and 6 for the PG stimulated TNF: IL-10. Interleukin-10, IL-6, and TNF production was not associated with white blood cell count, neutrophil count, and monocyte count. 3.3. Phagocytosis and respiratory burst Two samples were excluded from the control group from analysis of phagocytosis and respiratory burst, and two and four samples from the lymphoma group were excluded from phagocytosis and respiratory burst analysis respectively due to a technical problem with sample processing. Unstimulated cells (negative controls) in all groups had <1% positive cells. Control dogs had no difference in percentage of PMNs that performed phagocytosis of E. coli between week 1 and week 6, and no difference in percentage of PMNs undergoing E. coli or PMA-stimulated respiratory burst between week 1 and week 6. There was no significant difference at baseline between control dogs and dogs with lymphoma in percentage of cells performing phagocytosis of E. coli or E. coli or PMA-stimulated respiratory burst (data not shown).
Dogs with lymphoma had a significant decrease in percentage of E. coli-stimulated respiratory burst at week 6 compared with week 1 (Fig. 4, p = 0.016). There was no difference in E. coli phagocytosis or PMA stimulated respiratory burst between week 1 and 6 in dogs with lymphoma. When comparing dogs in complete remission with dogs in partial remission at week 6, remission status had no effect on phagocytosis or respiratory burst function (Table 3). 4. Discussion and conclusion We found that dogs with lymphoma had blunted leukocyte TNF production following stimulation with LPS, increased IL-10 production following PG stimulation, and decreased LPS and PG induced TNF: IL-10 production ratio when compared with control dogs. Following induction of remission there was no improvement in proand anti-inflammatory cytokine production imbalance. Of note is that the number of white blood cells had no effect on the amount of cytokines produced following maximal stimulation in this study in control dogs or dogs with lymphoma. In the whole blood culture
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Fig. 4. Comparison of the percentage of cells undergoing E. coli-induced respiratory burst from healthy dogs at baseline and dogs with lymphoma at baseline and week 6 of chemotherapy. See Fig. 2 for rest of key. b Samples are significantly different between week 1 and week 6 within the lymphoma group.
method employed in this study, blood volume (not the number of cells) is held constant in these cultures. We are therefore presenting data indicating whole blood production of cytokines as the measure of immune cell function. The total cytokine production (rather than cytokine production on a per-cell basis) is more relevant in this study in terms of overall assessment of clinical immunity in the dog. Additionally, chemotherapy decreased E. coli-induced respiratory burst function. We chose week 6 because all dogs, at this time point, had completed one full cycle of the chemotherapy protocol, had at least 2 weeks to recover from chemotherapy induced myelosuppression, and at least a 1 week wash-out of prednisone. Therefore, the direct impact of chemotherapy on immune function was minimized. These altered immune responses could have important implications in the management of canine lymphoma and reveal potential mechanisms for the increased risk of sepsis in dogs with lymphoma. We found that dogs with lymphoma also have diminished leukocyte TNF production capacity compared with control dogs following LPS stimulation and TNF production capacity did not improve following induction of remission. In humans, decreased TNF production capacity leads to worse outcomes in intensive care patients because a reduction in TNF results in increased susceptibility to microbial infection (Heagy et al., 2000, 2003). In addition, decreased LPS-induced TNF production has been documented in humans with haematopoietic neoplasia (Kaminska et al., 2001). There are multiple mechanisms that could explain altered leukocyte cytokine production in the lymphoma group including upregulation of IL-10, changes in cell surface receptors such as toll like receptor (TLR) 4, intracellular signalling cascade, mRNA translation and cytokine protein secretion (Baseggio et al., 2001; Hamoudi et al., 2010; Wolska et al., 2009) . It is possible that decreased TNF production capacity, and the lack of improvement following remission, may affect susceptibility to microbial infection. Knowledge of which dogs may be more susceptible to infection can identify dogs that cannot tolerate chemotherapy dose intensification and perhaps benefit from prophylactic antibiotic therapy following chemotherapy treatment. Interleukin-10 is an inhibitory cytokine, and its main role is to down regulate the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on activated macrophages and control innate immune reactions and cell mediated immunity.
Upregulation of IL-10 is a mechanism of immunodysfunction during critical illness in humans and increases morbidity in humans with sepsis (Muehlstedt et al., 2002; Muenzer et al., 2010). We found that dogs with lymphoma had an increase in IL-10 production following stimulation with PG prior to and during chemotherapy treatment. Further, we found that the ratio of LPS and PG induced TNF: IL-10 was decreased in dogs with lymphoma prior to treatment. This imbalance following LPS stimulation improved following chemotherapy, while the PG induced pro- and anti-inflammatory balance did not. Over production of interleukin-10 might contribute to decreased host response to microbial invasion and lead to an increased risk of infection. This is another factor identified in this study that may aid in selective chemotherapy dose intensification and alteration of chemotherapy protocols to prevent sepsis in dogs with lymphoma. Furthermore, the improvement in some areas but lack of change in others following induction of remission suggests that dogs with lymphoma may continue to have an increased risk of sepsis, regardless of remission status. Interleukin-6 functions in innate and adaptive immunity and stimulates the synthesis of acute phase proteins in addition to stimulating production of neutrophils by bone marrow progenitors (Candido and Hagemann, 2013). There was no significant difference between control dogs and dogs with lymphoma, baseline or week 6 in the lymphoma group in IL-6 production capacity in this study. Additionally, there were no differences in IL-6 production between dogs with partial and complete remission at week 6. Our findings are similar to those in humans with acute lymphocytic leukaemia (Kaminska et al., 2001) and may be due, in part, to the heterogeneity of T cell populations and their pattern of cytokine production. While TNF is mainly produced by macrophages and Th1 lymphocytes, IL-6 is produced by macrophages and Th2 lymphocytes. The differential decrease in pro-inflammatory cytokine production seen in this study may represent differences in effector reactions indicative of a decreased Th1 response and unchanged Th2 response. E. coli stimulated respiratory burst function was similar between dogs with lymphoma and controls prior to chemotherapy. However, after 6 weeks of chemotherapy, respiratory burst function was reduced in the lymphoma group. This is similar to that reported in dogs with lymphoma after 1 week of treatment (Leblanc et al., 2013), but is in contrast to data in humans showing suppressed
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respiratory burst function existed prior to therapy and partially improved following induction chemotherapy in acute myelogenous leukaemia patients (Hofmann et al., 1998). In children with acute lymphoblastic leukaemia, suppressed respiratory burst function was present both prior to and following induction chemotherapy (Tanaka et al., 2009). The decline in respiratory burst function in dogs could be attributed to direct immunosuppressive effects of chemotherapy as has been documented in humans (Mikirova et al., 2008); however, dogs had not received chemotherapy for at least 2 weeks prior to sample collection at the second time point. Also of interest is that respiratory burst function following PMA stimulation was not different than controls before chemotherapy and did not change during chemotherapy in dogs with lymphoma. As a protein kinase C ligand, PMA has been shown to activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase resulting in strong stimulation of respiratory burst. In contrast, E. coli mediated respiratory burst requires stimuli such as interferon-γ and signalling from TLRs. Since phagocytic function and PMA-stimulated respiratory burst were not appreciably altered in dogs with lymphoma, our results suggest chemotherapy induced dysfunction in TLRs or IFNγ production upstream of NADPH are potential pathways involved. Since the mechanisms behind the alterations in respiratory burst in dogs with lymphoma following chemotherapy remain unclear, further investigation of alterations in respiratory burst, including TLR function and IFNγ production are indicated. In humans with leukaemia, neutropaenia and impaired neutrophil function have both been considered risk factors for sepsis (Hubel et al., 1999). The mechanisms of suppressed neutrophil function in leukaemia patients were uncertain, although induction of T regulatory cells through increased IL-10 production was thought to be one mechanism by which neutrophil function was decreased (Hubel et al., 1999). Increased numbers of T regulatory cells have been noted in other studies of dogs with lymphoma (Mitchell et al., 2012; O’Neill et al., 2009). While T regulatory cells were not measured in this study, dogs with lymphoma did have an increased IL-10 production capacity which could result in induction of T regulatory cells and suppression of PMN function. Although we did not detect a difference in PMN phagocytosis or PMA stimulated respiratory burst, it is important to note that during chemotherapy, even with expected neutrophil and monocyte counts in circulation, the ability of these PMNs to perform respiratory burst when stimulated by E. coli was decreased which could lead to reduced microbial killing and an increased risk of infection. This study revealed that certain facets of pre-existing innate immune system dysfunction in dogs is not reversed with chemotherapy induced remission. In fact, dogs with lymphoma may be more susceptible to microbial infection despite normal numbers of circulating white blood cells and neutrophils. This immunodysfunction may help identify dogs at high risk of sepsis that would benefit from prophylactic antibiotic treatment during chemotherapy and those dogs that may tolerate dose intensification of chemotherapy. This investigation analysed the capacity of leukocytes to produce cytokines in response to stimulation with PAMPs in addition to the ability of PMNs to perform phagocytosis and respiratory burst at diagnosis and following induction chemotherapy in dogs with lymphoma. We found that dogs with lymphoma have blunted proinflammatory immune responses and increased anti-inflammatory responses demonstrated by a reduced capacity for leukocytes to produce TNF, increased IL-10 production, and decreased ratios of TNF to IL-10. Furthermore, dogs with lymphoma have decreased E. coli stimulated respiratory burst function following chemotherapy. Therefore, these data suggest that dogs with lymphoma had underlying immunodysfunction that may increase their risk of chemotherapy induced side effects. While chemotherapy dose intensification may improve the overall prognosis in dogs with
lymphoma, it will be imperative to fully understand the capacity of the immune system to respond to pathogens at diagnosis and during chemotherapy prior to dose intensification. This will allow targeted immunotherapy with chemotherapy for selected dose intensification without increases in morbidity associated with treatment.
Acknowledgements The authors would like to acknowledge the assistance of Anastasia Glahn, Matt Haight, and Deborah Tate in blood collection, and Drs. Carolyn Henry, Kim Selting, and Jeff Bryan for assistance in recruitment of dogs with lymphoma. This project was funded, in part, by an Acorn grant through the American Kennel Club Canine Health Foundation (01524-A).
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Mikirova, N.A., Klykov, A.A., Jackson, J.A., Riordan, N.H., 2008. Granulocyte activity in patients with cancer and healthy subjects. Cancer Biology and Therapeutics 7, 1362–1367. Mitchell, L., Dow, S.W., Slansky, J.E., Biller, B.J., 2012. Induction of remission results in spontaneous enhancement of anti-tumour cytotoxic T-lymphocyte activity in dogs with B cell lymphoma. Veterinary Immunology and Immunopathology 145, 597–603. Muehlstedt, S.G., Lyte, M., Rodriguez, J.L., 2002. Increased IL-10 production and HLA-DR suppression in the lungs of injured patients precede the development of nosocomial pneumonia. Shock (Augusta, Ga.) 17, 443–450. Muenzer, J.T., Davis, C.G., Chang, K., Schmidt, R.E., Dunne, W.M., Coopersmith, C.M., et al., 2010. Characterization and modulation of the immunosuppressive phase of sepsis. Infection and Immunity 78, 1582–1592. O’Neill, K., Guth, A., Biller, B., Elmslie, R., Dow, S., 2009. Changes in regulatory T cells in dogs with cancer and associations with tumour type. Journal of Veterinary Internal Medicine 23, 875–881. Pettengell, R., Schwenkglenks, M., Bosly, A., 2008. Association of reduced relative dose intensity and survival in lymphoma patients receiving CHOP-21 chemotherapy. Annals of Hematology 87, 429–430.
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Sorenmo, K.U., Harwood, L.P., King, L.G., Drobatz, K.J., 2010. Case-control study to evaluate risk factors for the development of sepsis (neutropenia and fever) in dogs receiving chemotherapy. Journal of the American Veterinary Medical Association 236, 650–656. Tanaka, F., Goto, H., Yokosuka, T., Yanagimachi, M., Kajiwara, R., Naruto, T., et al., 2009. Suppressed neutrophil function in children with acute lymphoblastic leukemia. International Journal of Hematology 90, 311–317. Vail, D.M., Pinkerton, M.E., Young, K.M., 2013. Canine lymphoma and lymphoid leukemias. In: Withrow, S.J., Vail, D.M., and Page, R.L. (Eds.), Small Animal Clinical Oncology, fifth ed. Elsevier, St. Louis, MO, pp. 608–638. Vaughan, A., Johnson, J.L., Williams, L.E., 2007. Impact of chemotherapeutic dose intensity and hematologic toxicity on first remission duration in dogs with lymphoma treated with a chemoradiotherapy protocol. Journal of Veterinary Internal Medicine 21, 1332–1339. Wolska, A., Lech-Maranda, E., Robak, T., 2009. Toll-like receptors and their role in hematologic malignancies. Current Molecular Medicine 9, 324–335.
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Chemotherapy and remission status do not alter pre-existing innate immune dysfunction in dogs with lymphoma S. Axiak-Bechtel a, B. Fowler b, D.H. Yu c, J. Amorim b, K. Tsuruta b, A. DeClue b,* a Comparative Oncology and Epigenetics Laboratory, Department of Veterinary Medicine and Surgery, University of Missouri, 900 East Campus Drive, Columbia, MO 65211, USA b Comparative Internal Medicine Laboratory, Department of Veterinary Medicine and Surgery, University of Missouri, 900 East Campus Drive, Columbia, MO 65211, USA c College of Veterinary Medicine, Chonnam National University, 77 Yongbong-ro, Buk-gu, 17 Gwangju, Korea
A R T I C L E
I N F O
Article history: Received 12 February 2014 Accepted 20 July 2014 Keywords: Innate immunity Lymphoma Chemotherapy Neutrophils
A B S T R A C T
Dogs with lymphoma have altered innate immunity and little is known about the effects of chemotherapy on innate immune function in dogs. Lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PG) – induced leukocyte cytokine production capacity, and phagocytosis and respiratory burst were evaluated in dogs prior to and following 6 weeks of chemotherapy. Dogs had decreased TNF production following LPS stimulation and increased IL-10 production following PG stimulation, which did not improve following remission of lymphoma. Dogs also had reduced E. coli-induced respiratory burst function after chemotherapy induced complete or partial remission. Dogs with lymphoma have an imbalance in proand anti-inflammatory cytokine production which did not improve with remission, and, following treatment, a decrease in respiratory burst function. Altered immune responses following exposure to bacterial pathogen associated molecular pattern motifs and bacteria may have many implications in the management of canine lymphoma. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lymphoma is the most common hematopoietic tumours in dogs (Dorn et al., 1970; Vail and Young, 2013). Despite the high initial response rate to chemotherapy, almost all affected dogs eventually relapse and ultimately die of lymphoma (Garrett et al., 2002; Vail and Young, 2013). The most common dose limiting adverse effect of chemotherapy is myelosuppression and subsequent immunosuppression, leading to increased risk of infection (Britton et al., 2012; Garrett et al., 2002; Vail and Young, 2013; Vaughan et al., 2007). Since quality of life is an important factor in determining treatment protocols for dogs, concerns with increased risk of infection have led to reductions of chemotherapy dose intensity. As a result, dogs receive lower than maximally tolerated doses of chemotherapy (Vaughan et al., 2007). Compounding these factors, dogs with lymphoma have an increased risk of developing sepsis with chemotherapy compared with dogs with other tumour types (Sorenmo et al., 2010). In humans, a chemotherapy dose reduction of 10% results in significantly reduced survival times (Pettengell et al., 2008). Similarly, dogs receiving maximum tolerated doses of chemotherapy as
* Corresponding author. Tel.: +1 573 882-7821; fax: +1 573 884-7563. E-mail address: [email protected] (A. DeClue). http://dx.doi.org/10.1016/j.rvsc.2014.07.009 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
indicated by significant myelosuppression (neutropenia) have a longer remission duration compared with dogs that do not experience myelosuppression (Vaughan et al., 2007). This implies that selective dose intensification may be associated with better outcome. However, increasing chemotherapy dose intensity in dogs may also lead to increased risk of sepsis and a reduction in quality of life, with chemotherapy induced sepsis having an 8.5% mortality rate (Britton et al., 2012). In addition to decreasing the number of phagocytic cells available to fight infection, induction chemotherapy will decrease the percentage of neutrophils capable of performing oxidative burst compared with pre-treatment (Leblanc et al., 2013); dogs with lymphoma also have an impaired ability to produce inflammatory cytokines in response to pathogen associated molecular patterns (PAMPs) (Fowler et al., 2011). These studies indicate that greater understanding of preexisting and chemotherapy-induced immunodysfunction is needed in dogs with lymphoma prior to pursuing dose intensification. Our objective was to investigate the effect of chemotherapy on leukocyte cytokine production capacity and circulating polymorphonuclear cell (PMN) phagocytosis and respiratory burst function. We hypothesized that dogs with lymphoma would have blunted leukocyte cytokine production capacity, PMN phagocytosis, and PMN respiratory burst compared with control dogs and that these immune function parameters would improve following chemotherapy induced remission.
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2. Materials and methods 2.1. Patient population This project was approved by the University of Missouri Animal Care and Use Committee (protocol #7334). Client owned dogs presenting to the University of Missouri, Veterinary Medical Teaching Hospital with a cytologic or histopathologic diagnosis of lymphoma were eligible for enrolment with informed owner consent. All dogs had a history, physical examination, and complete blood count (CBC) as part of routine diagnostics and staging. Other staging procedures routinely recommended, but not required, for dogs with lymphoma in this study included a biochemical profile, urinalysis, chest radiographs, abdominal ultrasound, bone marrow aspirate, and immunophenotyping. Dogs whose owners elected to use a standard of care, a six-month combination chemotherapy protocol (Garrett et al., 2002), were eligible for enrolment (Table 1). Following chemotherapy treatment, remission status was determined based on physical examination and CBC. The healthy control group consisted of dogs belonging to faculty, staff, and students at the University of Missouri. Dogs were deemed healthy based on history, physical examination, complete blood count, and plasma biochemical analysis. Dogs were staged based on the World Health Organization’s clinical staging system for lymphoma in domestic species. Dogs with a history of immunosuppressive therapy (including glucocorticoids, chemotherapy, or radiation therapy) in the month prior to presentation were excluded from the study. Additional exclusion criteria were previously diagnosed immune system disorder or history of inflammatory disease within three months of presentation. Dogs in the lymphoma group that failed to complete the first five treatments of chemotherapy were also excluded. Blood was collected into potassium EDTA, lithium heparin, and sodium heparin tubes for a CBC, biochemical profile, and immunologic evaluation, respectively. Dogs with lymphoma had blood collected at presentation prior to treatment (baseline) and again at week 6 of chemotherapy, prior to chemotherapy administration. Week 6 was defined as the week dogs were scheduled to begin the second cycle of treatment with vincristine. Therefore, this was at least 2 weeks after the first doxorubicin treatment and at least 1 week after discontinuation of prednisone. Remission status was recorded and based on physical examination findings and complete blood counts. Collection from control dogs occurred at baseline and again 6 weeks later to determine if time related fluctuations in immune parameters occurred. Samples for CBC, plasma biochemical profile and phagocytic function were processed within 12 hours of collection, and samples for cytokine evaluations processed within 2 h of collection. 2.2. Leukocyte cytokine production capacity Leukocyte cytokine production was performed as previously described (DeClue et al., 2008; Deitschel et al., 2010; Fowler et al., 2011). Five millilitres of whole blood was diluted 1:2 with complete Roswell Park Memorial Institute medium containing 200 U/ml of
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penicillin, 200 mg/ml of streptomycin (Gibco®, Invitrogen, Grand Island, NY, USA), placed in 12-well plates, and stimulated with the following pathogen associated molecular pattern motifs: lipopolysaccharide (LPS) from Escherichia coli (E. coli) O127:B8 (final well concentration, 100 ng/ml; Sigma-Aldrich, St. Louis, MO, USA), lipotechoic acid (LTA) from Streptococcus faecalis (final well concentration, 1000 ng/ml; Sigma-Aldrich), peptidoglycan (PG) from Staphylococcus aureus (final well concentration, 1000 ng/ml; SigmaAldrich), or control phosphate buffered saline (PBS). Wells were gently mixed on a plate rocker for 5 min, then incubated for 24 h at 37 °C and 5% CO2. The supernatant was then collected and stored at −80 °C until analysis. Tumour necrosis factor (TNF), interleukin (IL)-6, and IL-10 were measured in the supernatant using a canine specific multiplex bead based assay (Millipore, Billerica, MA, USA) (Karlsson et al., 2012). 2.3. Phagocytosis and respiratory burst function Granulocyte phagocytic and respiratory burst function tests were determined using commercially available test kits (Phagotest® and Phagoburst®, Orpegen Pharma, Heidelberg, Germany) previously validated for dogs (LeBlanc et al., 2010). For phagocytic function tests, 100 μl of whole blood was incubated with either 20 μl of washing solution (negative control) or opsonized, FITC-labelled E. coli (1 × 109 bacterial per ml) for 10 min, and extracellular fluorescence was removed by adding 100 μl of quenching solution (1×). Samples were washed using 3 ml of washing solution three times followed by incubation with 2 ml of fixing solution for red blood cell lysis and cell fixation. Granulocytes with phagocytized E. coli were measured by flow cytometry after DNA staining using propidium iodide. For the measurement of oxidative burst, 100 μl of whole blood was incubated with 20 μl of washing solution (negative control), opsonized E. coli (1 × 109 bacterial per ml), or phorbol myristate acetate (PMA, 0.0081 mM) for 10 min. Formation of reactive oxidants during oxidative burst was monitored by the addition and oxidation of 20 μl dihydrorhodamine 123 to Rhodamine 123 (1× solution). The reaction was stopped by addition of 2 ml of fixing and RBC lysing solution. Samples were then washed using 3 ml of washing solution. A DNA staining solution (propidium iodide, 1× solution) was then added and oxidative burst capacity was determined using flow cytometry. 2.4. Flow cytometry Flow cytometry was performed at the University of Missouri Cell and Immunology Core Facility using the CyAn ADP machine and summit software. DNA positive cells were identified on an FL2 histogram plot. These cells were then applied to a forward vs side scatter plot. Then granulocytes were identified and applied to an FL1histogram (Fig. 1). Percentage of FITC positive granulocytes having performed phagocytosis was recorded for assessment of phagocytic function. The percentage of Rhodamine 123 positive granulocytes having produced reactive oxygen metabolites was recorded for assessment of respiratory burst function. A minimum of 10,000 total events were recorded for each sample. 2.5. Statistical analysis
Table 1 Chemotherapy protocol used for dogs in this study. Week
0
L-asparaginase (400 IU/kg) Vincristine (0.5–0.7 mg/m2) Cyclophosphamide (200–250 mg/m2) Doxorubicin (1 mg/kg if <15 kg or 30 mg/m2 if >15 kg) Prednisone (tapering, starting at 2 mg/kg/day )
X
1
2
X
3
4
X X
X
X
X
6 X
X
X
5
X
Statistical analysis was performed using commercially available software (SigmaStat, Systat Software Inc.). Data distribution properties were tested using histogram plots. Differences in white blood cell count between the lymphoma and control groups at week 1 and 6 and between weeks in the lymphoma group were compared using two way repeated measures ANOVA and Fisher LSD method. Difference in variables between week 1 and week 6 in the control and lymphoma groups were compared using a Wilcoxon
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Fig. 1. Gating scheme for flow cytometry. First PI positive cells were identified, gated, and applied to a forward vs side scatter plot. Then a gate was drawn around neutrophils on the forward vs side scatter plot and applied to a FITC histogram.
signed rank test. Variables for dogs that achieved complete remission versus partial remission at week 6 in the lymphoma group were compared using a Mann–Whitney rank sum test. Multiple linear regression was used to evaluate the association among white blood cell count, neutrophil count, monocyte count, and cytokine production. A P-value of < 0.05 was considered statistically significant. Outliers were defined as any value greater than 1.5 times the interquartile range above or below the first or third quartile and were excluded from graphical presentation. 3. Results 3.1. Patient population Eleven control dogs were enrolled. Ages ranged from 2 to 10 years and the median age was seven years. Breeds enrolled were Australian Shepherd (n = 3), Labrador retriever (n = 3), Border collie (n = 2), and one each of German Shepherd Dog, Newfoundland, and Golden retriever. Weight ranged from 20 to 45 kg with a median of 30.5 kg. No noteworthy abnormalities were present on CBC or biochemical profile in any dog. Seventeen dogs with lymphoma were enrolled. Four dogs were excluded because they did not complete required treatment (n = 3) or they died prior to week 6 (n = 1). The ages of the remaining 13 dogs ranged from 4 to 14 years old and the median was eight years. Breeds included Beagle (n = 2), Labrador retriever (n = 2), mixed breed (n = 2) and one each of Rottweiler, Cocker Spaniel, Bull Terrier, Australian Shepherd, Golden Retriever, Border collie, and Schnauzer. Weight ranged from 10.8 to 49.2 kg and the median weight was 28.25 kg. Eleven dogs were staged with a CBC, chemistry panel, urinalysis, chest radiographs, abdominal ultrasound, and bone marrow
aspirate. All 11 of these dogs were immunophenotyped as B cell lymphoma using histopathology and immunohistochemistry. These dogs were considered stage IIIa (n = 3), IVa (n = 3), IVb (n = 2), Va (n = 1), and Vb (n = 2). Two of the 13 dogs were staged with CBC, chemistry panel, urinalysis, chest radiographs, and abdominal ultrasound, but no bone marrow aspirate. Both of these dogs were considered stage Va based on circulating lymphoblasts found on CBC. One dog’s immunophenotyping was consistent with B cell lymphoma and the other dog was not immunophenotyped because this diagnostic was declined by the pet owner. At the time of second blood sample collection, all dogs had received, in this order, L-asparaginase, prednisone, vincristine, cyclophosphamide, vincristine, and doxorubicin. The sample was collected at least 2 weeks after doxorubicin and at least 1 week after discontinuation of prednisone. Because some dog owners travelled long distances for chemotherapy appointments, dogs that were neutropaenic on a referring veterinarian CBC had treatment, and therefore their week 6 appointment, delayed by 1 week. Therefore, no dog was neutropaenic at the time of second sample collection. Six of the 13 dogs with lymphoma were in complete remission at the time of second sample collection, and seven dogs were in partial remission based on examination and CBC. The median and ranges of total white blood cell, neutrophil, monocyte, and lymphocyte counts are shown in Table 2. Dogs with lymphoma had a higher total white blood cell count and neutrophil count than control dogs at week 1 and 6. 3.2. Stimulated leukocyte cytokine production capacity In control dogs, no significant difference was present between week 1 and week 6 leukocyte production of: IL-6 when
Table 2 White blood cell, neutrophil, monocyte and lymphocyte counts for normal dogs and dogs with lymphoma, mean and standard deviation. Total white blood cells (cells/μl) Lymphoma week 1 Mean (standard deviation) Control week 1 Mean (standard deviation) Lymphoma week 6 Mean (standard deviation) Control week 6 Mean (standard deviation) a b
12,278 (5,873)
a
7050 (1,463) 11,350 (4,523) 7844 (2,839)
P ≤ 0.01 compared with control group during same week. P < 0.05 comparing same disease group in different weeks.
Neutrophils (cells/μl)
Monocytes (cells/μl)
Lymphocytes (cells/μl)
429 (345)
2224 (3373)
4555 (982)
434 (190)
1940 (928)
8502 (3,474)a
782 (518)a,b
1346 (615)
4846 (1,625)
340 (350)
1432 (723)
9560
(8,683)a
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Fig. 2. Comparison of leukocyte lipopolysaccharide-induced TNF production (A) and peptidoglycan-induced IL-10 production (B) from healthy dogs and dogs with lymphoma at week 1 (baseline) and week 6 of chemotherapy. Data are presented as a box and whisker plots. The upper and lower edges of the box represent the 75th and 25th percentiles respectively; the line within the box is the median value. Whiskers represent the range. a Baseline samples are significantly different between lymphoma and control groups at the same time point.
stimulated with LPS, LTA, or PG; IL-10 when stimulated with LPS, LTA, or PG; or TNF when stimulated with LPS, LTA, or PG. Compared with controls, dogs with lymphoma had blunted production of TNF following stimulation with LPS (p = 0.008; Fig. 2A). Dogs with lymphoma also produced significantly more IL-10 following stimulation with PG compared with control dogs (p = 0.015; Fig. 2B). There were no other differences in LPS, LTA, or PG induced TNF, IL-6 or IL-10 between control dogs and dogs with lymphoma. Lipopolysaccharide, LTA, or PG-induced leukocyte production of TNF, IL-6, or IL-10 was not significantly different between baseline and week 6 in the lymphoma group. Cytokine production at week 6 in dogs that achieved complete remission was not different that dogs that achieved only a partial remission (Table 3). At week 1, dogs with lymphoma had a significantly lower LPS induced TNF to IL-10 ratio (p = 0.022) and PG induced TNF: IL-10 ratio (p = 0.042) than control dogs (Fig. 3). When comparing these ratios between pre- and post-treatment in dogs with lymphoma, dogs treated with chemotherapy had a significantly higher LPS induced TNF: IL-10 ratio (p = 0.014; Fig. 3) when compared with baseline. No difference was found in TNF:IL-10 ratio for LTA
Table 3 Comparison of LPS, LTA and PG stimulated TNF, IL-6, IL-10 production and TNF:IL10 production ratio, phagocytosis of E. coli, E. coli-induced respiratory burst and PMAinduced respiratory burst between dogs with complete and partial remission after 6 weeks of chemotherapy. Data represented as median (Q1; Q3) in pg/ml (cytokines) or % cells (phagocytosis and respiratory burst). There were no differences in cytokine production between dogs in complete and partial remission. Parameter
Complete remission (n = 6)
Partial remission (n = 7)
LPS TNF LPS IL-6 LPS IL-10 LPS TNF:IL-10 LTA TNF LTA IL-6 LTA IL-10 LTA TNF:IL-10 PG TNF PG IL-6 PG IL-10 PG TNF:IL-10 E. coli phagocytosis E. coli respiratory burst PMA respiratory burst
1295 (565; 2303) 534 (258; 724) 1234 (516; 2418) 0.61 (5.7; 4.2) 1198 (785; 5278) 689 (345; 767) 1620 (553; 4484) 0.55 (0.34; 4.6) 791 (590; 2033) 407 (298; 666) 665 (473; 2303) 0.59 (0.4; 4.2) 91.2 (87.6; 94.3) 62.5 (40.6; 94.8) 97.3 (64.2; 99.3)
864 (697; 1295) 561 (320; 806) 979 (298; 5156) 1.1 (0.5; 2.3) 912 (874; 1299) 464 (348; 881) 1529 (500; 8141) 0.85 (0.31; 1.8) 734 (554; 917) 257 (217; 659) 681 (208; 7212) 1.29 (0.26; 1.68) 89.4 (82.6; 92.1) 69.5 (60.4; 88) 92.3 (67.3; 97.5)
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Fig. 3. Comparison of leukocyte lipopolysaccharide-induced TNF:IL-10 production ratios (A) and peptidoglycan-induced TNF:IL-10 production ratios (B) from healthy dogs and dogs with lymphoma at week 1 (baseline) and week 6 of chemotherapy. See Fig. 2 for rest of key. a Baseline samples are significantly different between lymphoma and control groups at the same time point. b Samples are significantly different between week 1 and week 6 within the lymphoma group.
stimulation or between week 1 and 6 for the PG stimulated TNF: IL-10. Interleukin-10, IL-6, and TNF production was not associated with white blood cell count, neutrophil count, and monocyte count. 3.3. Phagocytosis and respiratory burst Two samples were excluded from the control group from analysis of phagocytosis and respiratory burst, and two and four samples from the lymphoma group were excluded from phagocytosis and respiratory burst analysis respectively due to a technical problem with sample processing. Unstimulated cells (negative controls) in all groups had <1% positive cells. Control dogs had no difference in percentage of PMNs that performed phagocytosis of E. coli between week 1 and week 6, and no difference in percentage of PMNs undergoing E. coli or PMA-stimulated respiratory burst between week 1 and week 6. There was no significant difference at baseline between control dogs and dogs with lymphoma in percentage of cells performing phagocytosis of E. coli or E. coli or PMA-stimulated respiratory burst (data not shown).
Dogs with lymphoma had a significant decrease in percentage of E. coli-stimulated respiratory burst at week 6 compared with week 1 (Fig. 4, p = 0.016). There was no difference in E. coli phagocytosis or PMA stimulated respiratory burst between week 1 and 6 in dogs with lymphoma. When comparing dogs in complete remission with dogs in partial remission at week 6, remission status had no effect on phagocytosis or respiratory burst function (Table 3). 4. Discussion and conclusion We found that dogs with lymphoma had blunted leukocyte TNF production following stimulation with LPS, increased IL-10 production following PG stimulation, and decreased LPS and PG induced TNF: IL-10 production ratio when compared with control dogs. Following induction of remission there was no improvement in proand anti-inflammatory cytokine production imbalance. Of note is that the number of white blood cells had no effect on the amount of cytokines produced following maximal stimulation in this study in control dogs or dogs with lymphoma. In the whole blood culture
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Fig. 4. Comparison of the percentage of cells undergoing E. coli-induced respiratory burst from healthy dogs at baseline and dogs with lymphoma at baseline and week 6 of chemotherapy. See Fig. 2 for rest of key. b Samples are significantly different between week 1 and week 6 within the lymphoma group.
method employed in this study, blood volume (not the number of cells) is held constant in these cultures. We are therefore presenting data indicating whole blood production of cytokines as the measure of immune cell function. The total cytokine production (rather than cytokine production on a per-cell basis) is more relevant in this study in terms of overall assessment of clinical immunity in the dog. Additionally, chemotherapy decreased E. coli-induced respiratory burst function. We chose week 6 because all dogs, at this time point, had completed one full cycle of the chemotherapy protocol, had at least 2 weeks to recover from chemotherapy induced myelosuppression, and at least a 1 week wash-out of prednisone. Therefore, the direct impact of chemotherapy on immune function was minimized. These altered immune responses could have important implications in the management of canine lymphoma and reveal potential mechanisms for the increased risk of sepsis in dogs with lymphoma. We found that dogs with lymphoma also have diminished leukocyte TNF production capacity compared with control dogs following LPS stimulation and TNF production capacity did not improve following induction of remission. In humans, decreased TNF production capacity leads to worse outcomes in intensive care patients because a reduction in TNF results in increased susceptibility to microbial infection (Heagy et al., 2000, 2003). In addition, decreased LPS-induced TNF production has been documented in humans with haematopoietic neoplasia (Kaminska et al., 2001). There are multiple mechanisms that could explain altered leukocyte cytokine production in the lymphoma group including upregulation of IL-10, changes in cell surface receptors such as toll like receptor (TLR) 4, intracellular signalling cascade, mRNA translation and cytokine protein secretion (Baseggio et al., 2001; Hamoudi et al., 2010; Wolska et al., 2009) . It is possible that decreased TNF production capacity, and the lack of improvement following remission, may affect susceptibility to microbial infection. Knowledge of which dogs may be more susceptible to infection can identify dogs that cannot tolerate chemotherapy dose intensification and perhaps benefit from prophylactic antibiotic therapy following chemotherapy treatment. Interleukin-10 is an inhibitory cytokine, and its main role is to down regulate the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on activated macrophages and control innate immune reactions and cell mediated immunity.
Upregulation of IL-10 is a mechanism of immunodysfunction during critical illness in humans and increases morbidity in humans with sepsis (Muehlstedt et al., 2002; Muenzer et al., 2010). We found that dogs with lymphoma had an increase in IL-10 production following stimulation with PG prior to and during chemotherapy treatment. Further, we found that the ratio of LPS and PG induced TNF: IL-10 was decreased in dogs with lymphoma prior to treatment. This imbalance following LPS stimulation improved following chemotherapy, while the PG induced pro- and anti-inflammatory balance did not. Over production of interleukin-10 might contribute to decreased host response to microbial invasion and lead to an increased risk of infection. This is another factor identified in this study that may aid in selective chemotherapy dose intensification and alteration of chemotherapy protocols to prevent sepsis in dogs with lymphoma. Furthermore, the improvement in some areas but lack of change in others following induction of remission suggests that dogs with lymphoma may continue to have an increased risk of sepsis, regardless of remission status. Interleukin-6 functions in innate and adaptive immunity and stimulates the synthesis of acute phase proteins in addition to stimulating production of neutrophils by bone marrow progenitors (Candido and Hagemann, 2013). There was no significant difference between control dogs and dogs with lymphoma, baseline or week 6 in the lymphoma group in IL-6 production capacity in this study. Additionally, there were no differences in IL-6 production between dogs with partial and complete remission at week 6. Our findings are similar to those in humans with acute lymphocytic leukaemia (Kaminska et al., 2001) and may be due, in part, to the heterogeneity of T cell populations and their pattern of cytokine production. While TNF is mainly produced by macrophages and Th1 lymphocytes, IL-6 is produced by macrophages and Th2 lymphocytes. The differential decrease in pro-inflammatory cytokine production seen in this study may represent differences in effector reactions indicative of a decreased Th1 response and unchanged Th2 response. E. coli stimulated respiratory burst function was similar between dogs with lymphoma and controls prior to chemotherapy. However, after 6 weeks of chemotherapy, respiratory burst function was reduced in the lymphoma group. This is similar to that reported in dogs with lymphoma after 1 week of treatment (Leblanc et al., 2013), but is in contrast to data in humans showing suppressed
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respiratory burst function existed prior to therapy and partially improved following induction chemotherapy in acute myelogenous leukaemia patients (Hofmann et al., 1998). In children with acute lymphoblastic leukaemia, suppressed respiratory burst function was present both prior to and following induction chemotherapy (Tanaka et al., 2009). The decline in respiratory burst function in dogs could be attributed to direct immunosuppressive effects of chemotherapy as has been documented in humans (Mikirova et al., 2008); however, dogs had not received chemotherapy for at least 2 weeks prior to sample collection at the second time point. Also of interest is that respiratory burst function following PMA stimulation was not different than controls before chemotherapy and did not change during chemotherapy in dogs with lymphoma. As a protein kinase C ligand, PMA has been shown to activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase resulting in strong stimulation of respiratory burst. In contrast, E. coli mediated respiratory burst requires stimuli such as interferon-γ and signalling from TLRs. Since phagocytic function and PMA-stimulated respiratory burst were not appreciably altered in dogs with lymphoma, our results suggest chemotherapy induced dysfunction in TLRs or IFNγ production upstream of NADPH are potential pathways involved. Since the mechanisms behind the alterations in respiratory burst in dogs with lymphoma following chemotherapy remain unclear, further investigation of alterations in respiratory burst, including TLR function and IFNγ production are indicated. In humans with leukaemia, neutropaenia and impaired neutrophil function have both been considered risk factors for sepsis (Hubel et al., 1999). The mechanisms of suppressed neutrophil function in leukaemia patients were uncertain, although induction of T regulatory cells through increased IL-10 production was thought to be one mechanism by which neutrophil function was decreased (Hubel et al., 1999). Increased numbers of T regulatory cells have been noted in other studies of dogs with lymphoma (Mitchell et al., 2012; O’Neill et al., 2009). While T regulatory cells were not measured in this study, dogs with lymphoma did have an increased IL-10 production capacity which could result in induction of T regulatory cells and suppression of PMN function. Although we did not detect a difference in PMN phagocytosis or PMA stimulated respiratory burst, it is important to note that during chemotherapy, even with expected neutrophil and monocyte counts in circulation, the ability of these PMNs to perform respiratory burst when stimulated by E. coli was decreased which could lead to reduced microbial killing and an increased risk of infection. This study revealed that certain facets of pre-existing innate immune system dysfunction in dogs is not reversed with chemotherapy induced remission. In fact, dogs with lymphoma may be more susceptible to microbial infection despite normal numbers of circulating white blood cells and neutrophils. This immunodysfunction may help identify dogs at high risk of sepsis that would benefit from prophylactic antibiotic treatment during chemotherapy and those dogs that may tolerate dose intensification of chemotherapy. This investigation analysed the capacity of leukocytes to produce cytokines in response to stimulation with PAMPs in addition to the ability of PMNs to perform phagocytosis and respiratory burst at diagnosis and following induction chemotherapy in dogs with lymphoma. We found that dogs with lymphoma have blunted proinflammatory immune responses and increased anti-inflammatory responses demonstrated by a reduced capacity for leukocytes to produce TNF, increased IL-10 production, and decreased ratios of TNF to IL-10. Furthermore, dogs with lymphoma have decreased E. coli stimulated respiratory burst function following chemotherapy. Therefore, these data suggest that dogs with lymphoma had underlying immunodysfunction that may increase their risk of chemotherapy induced side effects. While chemotherapy dose intensification may improve the overall prognosis in dogs with
lymphoma, it will be imperative to fully understand the capacity of the immune system to respond to pathogens at diagnosis and during chemotherapy prior to dose intensification. This will allow targeted immunotherapy with chemotherapy for selected dose intensification without increases in morbidity associated with treatment.
Acknowledgements The authors would like to acknowledge the assistance of Anastasia Glahn, Matt Haight, and Deborah Tate in blood collection, and Drs. Carolyn Henry, Kim Selting, and Jeff Bryan for assistance in recruitment of dogs with lymphoma. This project was funded, in part, by an Acorn grant through the American Kennel Club Canine Health Foundation (01524-A).
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