James K. Belknap

Lamellar pro-inflammatory cytokine expression patterns in laminitis at the developmental stage and at the onset of lameness: innate vs. adaptive immune response.

REASONS FOR PERFORMING STUDY Recent research has indicated that inflammation plays a role in the early stages of laminitis and that, similar to organ failure in human sepsis, early inflammatory mechanisms may lead to downstream events resulting in lamellar failure. Characterisation of the type of immune response (i.e. innate vs. adaptive) is essential in order to develop therapeutic strategies to counteract these deleterious events. OBJECTIVES To quantitate gene expression of pro-inflammatory cytokines known to be important in the innate and adaptive immune response during the early stages of laminitis, using both the black walnut extract (BWE) and oligofructose (OF) models of laminitis. METHODS Real-time qPCR was used to assess lamellar mRNA expression of interleukins-1beta, 2, 4, 6, 8, 10, 12 and 18, and tumour necrosis factor alpha and interferon gamma at the developmental stage and at the onset of lameness. RESULTS Significantly increased lamellar mRNA expression of cytokines important in the innate immune response were present at the developmental stage of the BWE model, and at the onset of acute lameness in both the BWE model and OF model. Of the cytokines characteristic of the Th1 and Th2 arms of the adaptive immune response, a mixed response was noted at the onset of acute lameness in the BWE model, whereas the response was skewed towards a Th1 response at the onset of lameness in the OF model. CONCLUSIONS Lamellar inflammation is characterised by strong innate immune response in the developmental stages of laminitis; and a mixture of innate and adaptive immune responses at the onset of lameness. POTENTIAL RELEVANCE These results indicate that anti-inflammatory treatment of early stage laminitis (and the horse at risk of laminitis) should include not only therapeutic drugs that address prostanoid activity, but should also address the marked increases in lamellar cytokine expression.

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis.

REASONS FOR PERFORMING STUDY There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis-related laminitis than the black walnut extract (BWE) model. OBJECTIVES To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. METHODS Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature>38.9°C (DEV group, n=8) or at onset of Obel grade 1 lameness (OG1 group, n=8). Control horses (CON group, n=8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time-quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. RESULTS Increased mRNA concentrations (P<0.05) for IL-1β, IL-6, IL-12p35, COX-2, E-selectin and ICAM-1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL-2, IL-4, IL-10, TNF-α, IFN-γ or COX-1 at either the DEV or OG1 time points. CONCLUSIONS There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. POTENTIAL RELEVANCE The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.

Sepsis―From human organ failure to laminar failure

The horse with gram negative sepsis is known to be at particular risk of succumbing to laminitis. This review summarizes recent evidence indicating that similar pathologic events relating to inflammatory injury occur in laminar failure in laminitis as occur in organ injury/failure in human sepsis. The discussion also points out some important differences between the laminae and target organs in human sepsis that impact the clinical nature of the disease.

Proinflammatory Cytokine and Chemokine Gene Expression Profiles in Subcutaneous and Visceral Adipose Tissue Depots of Insulin-Resistant and Insulin-Sensitive Light Breed Horses

BACKGROUND Insulin resistance has been associated with risk of laminitis in horses. Genes coding for proinflammatory cytokines and chemokines are expressed more in visceral adipose tissue than in subcutaneous adipose tissue of insulin-resistant (IR) humans and rodents. HYPOTHESIS/OBJECTIVES To investigate adipose depot-specific cytokine and chemokine gene expression in horses and its relationship to insulin sensitivity (SI). ANIMALS Eleven light breed mares. METHODS Animals were classified as IR (SI=0.58+/-0.31x10(-4) L/min/mU; n=5) or insulin sensitive (IS; SI=2.59+/-1.21x10(-4) L/min/mU; n=6) based on results of a frequently sampled intravenous glucose tolerance test. Omental, retroperitoneal, and mesocolonic fat was collected by ventral midline celiotomy; incisional nuchal ligament and tail head adipose tissue biopsy specimens were collected concurrently. The expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, plasminogen activator inhibitor-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) in each depot was measured by real-time quantitative polymerase chain reaction. Data were analyzed by 2-way analysis of variance for repeated measures (P<.05). RESULTS No differences in TNF-alpha, IL-1beta, IL-6, PAI-1, or MCP-1 mRNA concentrations were noted between IR and IS groups for each depot. Concentrations of mRNA coding for IL-1beta (P=.0005) and IL-6 (P=.004) were significantly higher in nuchal ligament adipose tissue than in other depots. CONCLUSIONS AND CLINICAL IMPORTANCE These data suggest that the nuchal ligament depot has unique biological behavior in the horse and is more likely to adopt an inflammatory phenotype than other depots examined. Visceral fat may not contribute to the pathogenesis of obesity-related disorders in the horse as in other species.

Calprotectin in Myeloid and Epithelial Cells of Laminae from Horses with Black Walnut Extract‐Induced Laminitis

BACKGROUND Laminar inflammation is one of the earliest events in equine laminitis. Calprotectin (CP), a Damage-Associated Molecular Pattern protein, is overexpressed in inflammatory conditions of human skin. HYPOTHESIS CP is overexpressed in the laminar epidermis of horses with black walnut extract (BWE)-induced laminitis. ANIMALS Twenty adult horses. METHODS Experimental study. Horses were allocated to one of 4 groups. BWE was administered to horses in 3 groups, which were sampled 1.5, 3, and 12 hours (LAM) later. CP was visualized by immunohistochemistry. Laminar leukocyte counts and intensity of laminar epithelial staining were scored for all animals and statistically analyzed. RESULTS Laminar epidermal CP signal was significantly increased (P= .02) at the LAM time point, compared with other groups. Rare leukocytes were detected in laminae with CP staining in CON group, but there were marked increases in number of leukocytes in BWE-treated groups (P= .003). Sequential hematoxylin and eosin staining demonstrated that the majority of CP-positive leukocytes were perivascular polymorphonuclear neutrophils (PMN) at each of the developmental time points. CP-positive PMN and mononuclear cells were detected in perivascular locations and close to the epidermal basement membrane in the LAM group. CONCLUSIONS AND CLINICAL IMPORTANCE CP expression in the laminar epidermis occurs after extravasation of leukocytes, indicating that leukocyte emigration might be an initiating factor in laminar epithelial stress and inflammation in BWE-induced laminitis. These results indicate a possible role of CP in laminitis pathophysiology and laminar failure.

Laminar xanthine oxidase, superoxide dismutase and catalase activities in the prodromal stage of black-walnut induced equine laminitis

UNLABELLED REASONS FOR STUDY: Xanthine oxidase (XO)-dependent production of superoxide anion and hydrogen peroxide, a characteristic of ischaemia-reperfusion injury, may contribute to the development of equine laminitis. OBJECTIVE To determine the levels of XO and antioxidant enzymes (catalase, superoxide dismutase [SOD]) in the digital laminae of normal horses (CON) and horses in the developmental stage of laminitis using the black walnut extract (BWE) model. METHODS Healthy horses (n = 12) were administered BWE (BWE group, n = 6), or water (CON group, n = 6) through a nasogastric tube. At the onset of leucopenia in the BWE-treated animals, all horses were anaesthetised, digital laminae and other samples collected rapidly and flash frozen, and the animals subjected to euthanasia. Extracts of the frozen tissues were assayed for the 2 conformational forms of xanthine: oxygen oxidoreductase (XOR), namely, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), as well as the antioxidant enzymes, SOD and catalase. RESULTS Extracts of liver, lungs and skin, but not digital laminae, from either CON or BWE-treated horses had endogenous SOD, whereas all had endogenous XO and catalase. The levels of XDH, XO and catalase were similar in extracts of laminae from CON and BWE-treated horses as was the ratio of XDH to XO in extracts. CONCLUSIONS AND POTENTIAL RELEVANCE The absence of increased XO activity suggest against the involvement of this reactive oxygen intermediate-generating system in the development of laminar pathology in BWE-treated horses. Conversely, the absence of SOD from extracts of equine digital laminae, but not other tissues, suggests that the equine digital laminae are highly susceptible to damage by superoxide anion, produced, for example, by emigrant inflammatory leucocytes.

Leukocyte-derived and endogenous matrix metalloproteinases in the lamellae of horses with naturally acquired and experimentally induced laminitis.

RATIONALE Inflammation and dysregulation of endogenous matrix metalloproteinase (MMP) production are implicated in the development of equine laminitis. In this study, we examine quantitative relationships among levels of leukocyte-derived proMMP-9 and MMP-9, lamellar proMMP-2 and MMP-2, and expression of proMMP-2 processing enzymes, MT1-MMP/PACE4, as steps towards determining whether inflammation and dysregulation of endogenous MMP production are independent or co-dependent processes. ANIMALS Archived samples of lamellae from horses with naturally acquired laminitis (n = 12), and from horses administered a pro-laminitic gastric bolus of starch gruel were used, the latter horses falling into two groups: (i) responders (CHO-R, n = 7), which developed Obel grade 3-lameness and (ii) non-responders (CHO-NR, n = 4), which did not become lame. METHODS Lamellar tissue extracts were analyzed by gelatin zymography to determine gelatinase content and by a myeloperoxidase ELISA to quantify relative monocyte/neutrophil content in the tissue. Real-time PCR was employed to measure gene expression of MT1-MMP and PACE4. RESULTS AND CONCLUSIONS Extracts of lamellae from control horses, CHO-NR and horses with chronic (non-aggravated) laminitis had similarly low levels of pro and processed MMP-9 and MMP-2. In contrast, proMMP-9 was significantly elevated in extracts of lamellae from CHO-R and horses with naturally acquired acute and aggravated chronic laminitis. Lamellar MMP-2 was also increased significantly in the CHO-R and aggravated chronic laminitis groups, although not in the horses with naturally acquired acute laminitis. Concentrations of proMMP-9 correlated directly with myeloperoxidase content in lamellar extracts, suggesting production/induction by inflammatory leukocytes. In contrast, concentrations of proMMP-2 and MMP-2 were unrelated to concentrations of myeloperoxidase or proMMP-9 suggesting that leukocyte infiltration and dysregulation of endogenous MMP-2 are independent processes most likely with distinct inducers. Neither MT1-MMP nor PACE4 gene expression was elevated relative to controls in any group; this is discussed with respect to proMMP-2 processing in disease. In addition, variability in relative concentrations of lamellar MMPs observed among horses with Obel grade 3-lameness is discussed in the context of laminitis risk assessment and disease outcome.

Digital hypothermia inhibits early lamellar inflammatory signalling in the oligofructose laminitis model.

REASONS FOR PERFORMING STUDY The pathophysiological events inhibited by prophylactic digital hypothermia that result in reduction of the severity of acute laminitis are unknown. OBJECTIVES To determine if digital hypothermia inhibits lamellar inflammatory signalling during development of oligofructose (OF) induced laminitis. METHODS Fourteen Standardbred horses were given 10 g/kg bwt OF by nasogastric tube with one forelimb (CRYO) continuously cooled by immersion in ice and water and one forelimb (NON-RX) at ambient temperature. Lamellae were harvested prior to the onset of lameness (24 h post OF administration, DEV group, n = 7) or at the onset of lameness (OG1 group, n = 7). Lamellar mRNA was purified and cDNA produced for real time-quantitative PCR analysis of mRNA concentrations of cytokines (IL-6, IL-1β, IL-10), chemokines (CXCL1, CXCL6, CXCL8/IL-8, MCP-1, MCP-2), cell adhesion molecules (ICAM-1, E-selectin), COX-2 and 3 housekeeping genes. Data were analysed (NON-RX vs. CRYO, NON-RX vs. archived control [CON, n = 7] lamellar tissue) using nonparametric tests. RESULTS Compared with CON, the OG1 NON-RX had increased (P<0.05) lamellar mRNA concentrations of all measured mediators except IL-10, IL-1β and MCP-1/2, whereas only CXCL8 was increased (P<0.05) in DEV NON-RX. Within the OG1 group, CRYO limbs (compared with NON-RX) had decreased (P<0.05) mRNA concentrations of the majority of measured inflammatory mediators (no change in MCP-1 and IL-10). Within the DEV group, mRNA concentrations of CXCL-1, ICAM-1, IL-1β, CXCL8 and MCP-2 were decreased (P<0.05) and the anti-inflammatory cytokine IL-10 was increased (compared with NON-RX limbs; P<0.05). CONCLUSIONS Digital hypothermia effectively blocked early lamellar inflammatory events likely to play an important role in lamellar injury including the expression of chemokines, proinflammatory cytokines, COX-2 and endothelial adhesion molecules. POTENTIAL RELEVANCE This study demonstrates a potential mechanism by which hypothermia reduces the severity of acute laminitis, and may help identify molecular targets for future laminitis intervention.

Insulin resistance selectively alters cell-surface glucose transporters but not their total protein expression in equine skeletal muscle.

BACKGROUND Insulin resistance (IR) has been widely recognized in humans, and more recently in horses, but its underlying mechanisms are still not well understood. The translocation of glucose transporter 4 (GLUT4) to the cell surface is the limiting step for glucose uptake in insulin-sensitive tissues. Although the downstream signaling pathways regulating GLUT translocation are not well defined, AS160 recently has emerged as a potential key component. In addition, the role of GLUT12, one of the most recently identified insulin-sensitive GLUTs, during IR is unknown. HYPOTHESIS/OBJECTIVES We hypothesized that cell-surface GLUT will be decreased in muscle by an AS160-dependent pathway in horses with IR. ANIMALS Insulin-sensitive (IS) or IR mares (n = 5/group). METHODS Muscle biopsies were performed in mares classified as IS or IR based on results of an insulin-modified frequently sampled IV glucose tolerance test. By an exofacial bis-mannose photolabeled method, we specifically quantified active cell-surface GLUT4 and GLUT12 transporters. Total GLUT4 and GLUT12 and AS160 protein expression were measured by Western blots. RESULTS IR decreased basal cell-surface GLUT4 expression (P= .027), but not GLUT12, by an AS160-independent pathway, without affecting total GLUT4 and GLUT12 content. Cell-surface GLUT4 was not further enhanced by insulin stimulation in either group. CONCLUSIONS AND CLINICAL IMPORTANCE IR induced defects in the skeletal muscle glucose transport pathway by decreasing active cell-surface GLUT4.

Cloning and expression of ADAM-related metalloproteases in equine laminitis.

Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic and aggravated chronic clinical cases) or experimentally-induced (black walnut extract (BWE) and starch gruel models) laminitis. Changes in expression of these enzymes and regulators may underlie the pathologic remodeling of lamellar tissue in laminitis. Genes encoding ADAMs involved in inflammation (ADAM-10 and ADAM-17), as well as those implicated in arthritis (ADAMTS-1, ADAMTS-4 and ADAMTS-5) were cloned, and the sequences used to generate specific oligonucleotide primers for the RT-qPCR experiments. Our results show that genes encoding ADAM-10 and ADAM-17 were not induced in most laminitic animals, whereas ADAMTS-4 gene expression was strongly upregulated in nearly all horses with experimentally-induced and naturally-acquired laminitis. The expression of matrix metalloproteases (MMP)-9 and ADAMTS-5 was also increased in many of the laminitic horses. In addition, TIMP-2 gene expression was decreased in most laminitic horses, whereas expression of genes encoding other TIMPs, namely TIMP-1 and TIMP-3, was randomly increased or decreased in the various models. We conclude that increased expression of lamellar ADAMTS-4 is a common feature of laminitis consistent with a central role of the gene product in the pathophysiology of the disease.