Impact of Equine Ocular Surface Squamous Neoplasia on Interactions between Ocular Transcriptome and Microbiome.

Ocular surface squamous neoplasia (OSSN) represents the most common conjunctival tumor in horses and frequently results in vision loss and surgical removal of the affected globe. Multiple etiologic factors have been identified as contributing to OSSN progression, including solar radiation exposure, genetic mutations, and a lack of periocular pigmentation. Response to conventional treatments has been highly variable, though our recent work indicates that these tumors are highly responsive to local immunotherapy. In the present study, we extended our investigation of OSSN in horses to better understand how the ocular transcriptome responds to the presence of the tumor and how the ocular surface microbiome may also be altered by the presence of cancer. Therefore, we collected swabs from the ventral conjunctival fornix from 22 eyes in this study (11 with cytologically or histologically confirmed OSSN and 11 healthy eyes from the same horses) and performed RNA sequencing and 16S microbial sequencing using the same samples. Microbial 16s DNA sequencing and bulk RNA sequencing were both conducted using an Illumina-based platform. In eyes with OSSN, we observed significantly upregulated expression of genes and pathways associated with inflammation, particularly interferon. Microbial diversity was significantly reduced in conjunctival swabs from horses with OSSN. We also performed interactome analysis and found that three bacterial taxa (Actinobacillus, Helcococcus and Parvimona) had significant correlations with more than 100 upregulated genes in samples from animals with OSSN. These findings highlight the inflammatory nature of OSSN in horses and provide important new insights into how the host ocular surface interacts with certain microbial populations. These findings suggest new strategies for the management of OSSN in horses, which may entail immunotherapy in combination with ocular surface probiotics or prebiotics to help normalize ocular cell and microbe interactions.

Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity.

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Intralesional interferon alpha-2b as a novel treatment for periocular squamous cell carcinoma in horses.

OBJECTIVE: To determine the safety and efficacy of perilesional human recombinant interferon alpha-2b (IFNα2b) for treatment of periocular squamous cell carcinoma (PSCC) in horses. ANIMALS STUDIED: Eleven horses (12 eyes) with PSCC were enrolled in this prospective clinical study with owner consent. PROCEDURES: Systemically healthy horses were included in the study following confirmation of PSCC via biopsy. Every two weeks for a maximum of six treatments, horses were sedated and perilesional injection of IFNα2b (10 million IU) was performed. Tumors were measured prior to each injection and at one, three, and 12 months after treatment completion. A greater than 50% reduction in tumor size was considered positive response to treatment (i.e., partial or complete response). Development of anti-IFNα2b antibodies was assessed using serum samples obtained after treatment initiation and compared with treatment responses. Antibody concentrations were analyzed using a mixed model. Statistical significance was considered p < 0.05. RESULTS: Each horse received four to six perilesional injections of IFNα2b. Five of 12 eyes (4/11 horses) responded to treatment. Two of five eyes showed complete resolution of gross PSCC. No systemic adverse effects were seen. Local swelling occurred during treatment protocol in 6/11 horses but resolved without intervention. All horses developed serum anti-IFNα2b antibodies. There was no evidence of statistical difference in antibody concentration between responders and non-responders. CONCLUSIONS: Perilesional administration of IFNα2b was found to be well-tolerated in horses with PSCC, and induced tumor regression in 42% of treated eyes. Treatment failure appears unrelated to the development of IFNα2b antibodies.

Direct comparison of canine and human immune responses using transcriptomic and functional analyses.

The canine spontaneous cancer model is increasingly utilized to evaluate new combined cancer immunotherapy approaches. While the major leukocyte subsets and phenotypes are closely related in dogs and humans, the functionality of T cells and antigen presenting cells in the two species has not been previously compared in detail. Such information would be important in interpreting immune response data and evaluating the potential toxicities of new cancer immunotherapies in dogs. To address this question, we used in vitro assays to compare the transcriptomic, cytokine, and proliferative responses of activated canine and human T cells, and also compared responses in activated macrophages. Transcriptomic analysis following T cell activation revealed shared expression of 515 significantly upregulated genes and 360 significantly downregulated immune genes. Pathway analysis identified 33 immune pathways shared between canine and human activated T cells, along with 34 immune pathways that were unique to each species. Activated human T cells exhibited a marked Th1 bias, whereas canine T cells were transcriptionally less active overall. Despite similar proliferative responses to activation, canine T cells produced significantly less IFN-γ than human T cells. Moreover, canine macrophages were significantly more responsive to activation by IFN-γ than human macrophages, as reflected by co-stimulatory molecule expression and TNF-α production. Thus, these studies revealed overall broad similarity in responses to immune activation between dogs and humans, but also uncovered important key quantitative and qualitative differences, particularly with respect to T cell responses, that should be considered in designing and evaluating cancer immunotherapy studies in dogs.

Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated mesenchymal stromal cells reveals induction of T-cell response pathways.

OBJECTIVE: To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology. ANIMALS: 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4). METHODS: Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints. RESULTS: Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20). CLINICAL RELEVANCE: Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.

Modulation of In Vitro Macrophage Responses via Primary and Secondary Bile Acids in Dogs.

Bile acids (BA) are important metabolites secreted into the intestinal lumen and impacted by luminal microbes and dietary intake. Prior studies in humans and rodents have shown that BAs are immunologically active and that primary and secondary BAs have distinct immune properties. Therefore, the composition of the gut BA pool may influence GI inflammatory responses. The current study investigated the relative immune modulatory properties of primary (cholic acid, CA) and secondary BAs (lithocholic acid, LCA) by assessing their effects on canine macrophage cytokine secretion and BA receptor (TGR5) expression. In addition, RNA sequencing was used to further interrogate how CA and LCA differentially modulated macrophage responses to LPS (lipopolysaccharide). We found that exposure to either CA or LCA influenced LPS-induced cytokine production via macrophages similarly, with suppression of TNF-α secretion and enhancement of IL-10 secretion. Neither BA altered the expression of the BA receptor TGR5. Transcriptomic analysis revealed that CA activated inflammatory signaling pathways in macrophages involving type II interferon signaling and the aryl hydrocarbon receptor, whereas LCA activated pathways related to nitric oxide signaling and cell cycle regulation. Thus, we concluded that both primary and secondary BAs are active modulators of macrophage responses in dogs, with differential and shared effects evident with sequencing analysis.

Efficacy of an elemental diet in achieving clinical remission in dogs with chronic enteropathy.

BACKGROUND: Diet may induce clinical remission in dogs with chronic enteropathy (CE). Elemental diets (EDs), providing protein as amino acids, modulate intestinal immunity and microbiome in rodents and humans. HYPOTHESIS: Evaluate the impact of an amino acid-based kibble (EL) on CE clinical activity and gastrointestinal (GI)-relevant variables. ANIMALS: Client-owned dogs (n = 23) with inadequately controlled CE. METHODS: Prospective, uncontrolled clinical trial. Diagnostic evaluation including upper and lower GI endoscopy was performed before study entry. Canine chronic enteropathy clinical activity index (CCECAI), serum biomarkers, and fecal microbiome were evaluated before and after 2 weeks of EL. Dogs with stable or improved CE remained in the study for another 6 weeks. Pre- and post-EL clinical and microbiological variables were compared statistically using a mixed model. RESULTS: After 2 weeks of EL, 15 of 22 dogs (68%; 95% confidence interval [CI], 47%-84%) consuming the diet were classified as responders with a median (range) decrease in CCECAI from 6 (3-12) to 2 (0-9; P < .001). Fourteen of 15 responders and 2/7 nonresponders at 2 weeks completed the trial; all 16 were experiencing adequate control at week 8 with a median CCECAI of 2 (0-3). In total, 16/23 dogs (70%; 95% CI, 49%-84%) were responders. Feeding EL caused shifts in fecal bacterial communities, which differed between responders and nonresponders. Serum biomarker concentrations were unchanged throughout the study apart from serum alkaline phosphatase activity. CONCLUSIONS: Exclusive feeding of EL improved clinical signs in 16 of 23 dogs with uncontrolled CE. Fecal microbiome shifts were associated with response to diet and may represent a mechanism for clinical improvement.

Erythrocyte removal from bone marrow aspirate concentrate improves efficacy as intra-articular cellular therapy in a rodent osteoarthritis model.

Background: Despite the high prevalence of osteoarthritis (OA), there remains a need for additional therapeutic options. Cellular therapies with minimally manipulated cells such as bone marrow aspirate concentrates (BMAC) are increasingly popular in the U.S. but clear-cut evidence of efficacy has not been established. In theory, BMAC injections provide a source of stromal cells to stimulate healing in OA and ligamentous injuries; however, BMAC injections are also often associated with inflammation, short-term pain, and mobility impairment. Given that blood is known to trigger inflammation in joints, we hypothesized that removing erythrocytes [red blood cells (RBCs)] from BMAC preparations prior to intra-articular injection would improve efficacy for OA treatment. Methods: To test this hypothesis, BMAC was collected from the bone marrow of mice. Three treatment groups were pursued: (I) untreated; (II) BMAC; or (III) BMAC depleted of RBCs by lysis. Product was injected into the femorotibial joint of mice 7 days after OA had been induced by destabilization of the medial meniscus (DMM). To assess the impact of treatment on joint function, individual cage monitoring (ANY-mazeTM) and Digigait treadmill-based analyses were performed over 4 weeks. At study completion, joint histopathology was assessed and immune transcriptomes within joint tissues were compared using a species-specific NanoString panel. Results: Significant improvements in activity, gait parameters, and histology scores were seen in animals receiving RBC-depleted BMAC compared to untreated mice; animals treated with non-depleted BMAC did not demonstrate this same extent of consistent significant improvement. Transcriptomic analysis of joint tissues revealed significant upregulation of key anti-inflammatory genes, including interleukin-1 receptor antagonist (IRAP), in mice treated with RBC-depleted BMAC compared to animals treated with non-RBC depleted BMAC. Conclusions: These findings indicate that RBC depletion in BMAC prior to intra-articular injection improves treatment efficacy and reduces joint inflammation compared to BMAC.

Antimicrobial Properties of Equine Stromal Cells and Platelets and Future Directions.

Increasing antimicrobial resistance in veterinary practice has driven the investigation of novel therapeutic strategies including regenerative and biologic therapies to treat bacterial infection. Integration of biological approaches such as platelet lysate and mesenchymal stromal cell (MSC) therapy may represent adjunctive treatment strategies for bacterial infections that minimize systemic side effects and local tissue toxicity associated with traditional antibiotics and that are not subject to antibiotic resistance. In this review, we will discuss mechanisms by which biological therapies exert antimicrobial effects, as well as potential applications and challenges in clinical implementation in equine practice.

Evaluation of Immune Nanoparticles for Rapid and Non-Specific Activation of Antiviral and Antibacterial Immune Responses in Cattle, Swine, and Poultry.

Given the rapid potential spread of agricultural pathogens, and the lack of vaccines for many, there is an important unmet need for strategies to induce rapid and non-specific immunity against these viral and bacterial threats. One approach to the problem is to generate non-specific immune responses at mucosal surfaces to rapidly protect from entry and replication of both viral and bacterial pathogens. Using complexes of charged nanoparticle liposomes with both antiviral and antibacterial toll-like receptor (TLR) nucleic acid ligands (termed liposome-TLR complexes or LTC), we have previously demonstrated considerable induction of innate immune responses in nasal and oropharyngeal tissues and protection from viral and bacterial pathogens in mixed challenge studies in rodents, cattle, and companion animals. Therefore, in the present study, we used in vitro assays to evaluate the ability of the LTC immune stimulant to activate key innate immune pathways, particularly interferon pathways, in cattle, swine, and poultry. We found that LTC complexes induced strong production of type I interferons (IFNα and IFNß) in both macrophages and leukocyte cultures from all three species. In addition, the LTC complexes induced the production of additional key protective cytokines (IL-6, IFNγ, and TNFα) in macrophages and leukocytes in cattle and poultry. These findings indicate that the LTC mucosal immunotherapeutic has the capability to activate key innate immune defenses in three major agricultural species and potentially induce broad protective immunity against both viral and bacterial pathogens. Additional animal challenge studies are warranted to evaluate the protective potential of LTC immunotherapy in cattle, swine, and poultry.

Strategies to overcome myeloid cell induced immune suppression in the tumor microenvironment.

Cancer progression and metastasis due to tumor immune evasion and drug resistance is strongly associated with immune suppressive cellular responses, particularly in the case of metastatic tumors. The myeloid cell component plays a key role within the tumor microenvironment (TME) and disrupts both adaptive and innate immune cell responses leading to loss of tumor control. Therefore, strategies to eliminate or modulate the myeloid cell compartment of the TME are increasingly attractive to non-specifically increase anti-tumoral immunity and enhance existing immunotherapies. This review covers current strategies targeting myeloid suppressor cells in the TME to enhance anti-tumoral immunity, including strategies that target chemokine receptors to deplete selected immune suppressive myeloid cells and relieve the inhibition imposed on the effector arms of adaptive immunity. Remodeling the TME can in turn improve the activity of other immunotherapies such as checkpoint blockade and adoptive T cell therapies in immunologically "cold" tumors. When possible, in this review, we have provided evidence and outcomes from recent or current clinical trials evaluating the effectiveness of the specific strategies used to target myeloid cells in the TME. The review seeks to provide a broad overview of how myeloid cell targeting can become a key foundational approach to an overall strategy for improving tumor responses to immunotherapy.

Distinct differences in immunological properties of equine orthobiologics revealed by functional and transcriptomic analysis using an activated macrophage readout system.

Introduction: Multiple biological therapies for orthopedic injuries are marketed to veterinarians, despite a lack of rigorous comparative biological activity data to guide informed decisions in selecting a most effective compound. Therefore, the goal of this study was to use relevant bioassay systems to directly compare the anti-inflammatory and immunomodulatory activity of three commonly used orthobiological therapies (OTs): mesenchymal stromal cells (MSC), autologous conditioned serum (ACS), and platelet rich plasma (PRP). Methods: Equine monocyte-derived macrophages were used as the readout system to compare therapies, including cytokine production and transcriptomic responses. Macrophages were stimulated with IL-1ß and treated 24 h with OTs, washed and cultured an additional 24 h to generate supernatants. Secreted cytokines were measured by multiplex immunoassay and ELISA. To assess global transcriptomic responses to treatments, RNA was extracted from macrophages and subjected to full RNA sequencing, using an Illumina-based platform. Data analysis included comparison of differentially expressed genes and pathway analysis in treated vs. untreated macrophages. Results: All treatments reduced production of IL-1ß by macrophages. Secretion of IL-10 was highest in MSC-CM treated macrophages, while PRP lysate and ACS resulted in greater downregulation of IL-6 and IP-10. Transcriptomic analysis revealed that ACS triggered multiple inflammatory response pathways in macrophages based on GSEA, while MSC generated significant downregulation of inflammatory pathways, and PRP lysate induced a mixed immune response profile. Key downregulated genes in MSC-treated cultures included type 1 and type 2 interferon response, TNF-α and IL-6. PRP lysate cultures demonstrated downregulation of inflammation-related genes IL-1RA, SLAMF9, ENSECAG00000022247 but concurrent upregulation of TNF-α, IL-2 signaling, and Myc targets. ACS induced upregulation of inflammatory IL-2 signaling, TNFα and KRAS signaling and hypoxia, but downregulation of MTOR signaling and type 1 interferon signaling. Discussion: These findings, representing the first comprehensive look at immune response pathways for popular equine OTs, reveal distinct differences between therapies. These studies address a critical gap in our understanding of the relative immunomodulatory properties of regenerative therapies commonly used in equine practice to treat musculoskeletal disease and will serve as a platform from which further in vivo comparisons may build.

Chronic adrenergic stress and generation of myeloid-derived suppressor cells: Implications for cancer immunotherapy in dogs.

Recent studies have highlighted a key role played by the sympathetic nervous system (SNS) and adrenergic stress in mediating immune suppression associated with chronic inflammation in cancer and other diseases. The connection between chronic SNS activation, adrenergic stress and immune suppression is linked in part to the ability of catecholamines to stimulate the bone marrow release and differentiation of myeloid-derived suppressor cells (MDSC). Rodent model studies have revealed an important role for ß-adrenergic receptor signalling in suppression of cancer immunity in mice subjected to chronic stresses, including thermal stress. Importantly, therapeutic blockade of beta-adrenergic responses by drugs such as propranolol can partially reverse the generation and differentiation of MDSC, and partly restore tumour immunity. Clinical trials in both humans and dogs with cancer have demonstrated that propranolol blockade can improve responses to radiation therapy, cancer vaccines and immune checkpoint inhibitors. Thus, the SNS stress response has become an important new target to relieve immune suppression in cancer and other chronic inflammatory conditions.

Nanoparticle ocular immunotherapy for herpesvirus surface eye infections evaluated in cat infection model.

Ocular herpes simplex type 1 (HSV-1) infections can trigger conjunctivitis, keratitis, uveitis, and occasionally retinitis, and is a major cause of blindness worldwide. The infections are lifelong and can often recrudesce during periods of stress or immune suppression. Currently HSV-1 infections of the eye are managed primarily with anti-viral eye drops, which require frequent administration, can cause irritation, and may take weeks for full resolution of symptoms. We therefore evaluated the effectiveness of an ocular immune activating nanoparticle eye drop as a novel approach to treating HSV-1 infection, using a cat feline herpesvirus -1 (FHV-1) ocular infection model. In vitro studies demonstrated significant induction of both type I and II interferon responses by the liposome-dual TLR 3/9 agonist nanoparticles, along with suppression of FHV-1 replication. In cats with naturally occurring eye infections either proven or suspected to involve FHV-1, ocular nanoparticle treated animals experienced resolution of signs within several days of treatment, including resolution of keratitis and corneal ulcers. In a cat model of recrudescent FHV-1 infection, cats treated twice daily with immune nanoparticle eye drops experienced significant lessening of ocular signs of infection and significantly fewer episodes of viral shedding compared to control cats. Treatment was well-tolerated by all cats, without signs of drug-induced ocular irritation. We concluded therefore that non-specific ocular immunotherapy offers significant promise as a novel approach to treatment of HSV-1 and FHV-1 ocular infections.

Novel ocular immunotherapy induces tumor regression in an equine model of ocular surface squamous neoplasia.

Ocular surface squamous neoplasia (OSSN) is the major cause of corneal cancer in man and horses worldwide, and the prevalence of OSSN is increasing due to greater UVB exposure globally. Currently, there are no approved treatments for OSSN in either species, and most patients are managed with surgical excision or off-label treatment with locally injected interferon alpha, or topically applied cytotoxic drugs such as mitomycin C. A more broadly effective and readily applied immunotherapy could exert a significant impact on management of OSSN worldwide. We therefore evaluated the effectiveness of a liposomal TLR complex (LTC) immunotherapy, which previously demonstrated strong antiviral activity in multiple animal models following mucosal application, for ocular antitumor activity in a horse spontaneous OSSN model. In vitro studies demonstrated strong activation of interferon responses in horse leukocytes by LTC and suppression of OSSN cell growth and migration. In a trial of 8 horses (9 eyes), treatment with topical or perilesional LTC resulted in an overall tumor response rate of 67%, including durable regression of large OSSN tumors. Repeated treatment with LTC ocular immunotherapy was also very well tolerated clinically. We conclude therefore that ocular immunotherapy with LTC warrants further investigation as a novel approach to management of OSSN in humans.

TLR-activated mesenchymal stromal cell therapy and antibiotics to treat multi-drug resistant Staphylococcal septic arthritis in an equine model.

Background: Rapid development of antibiotic resistance necessitates advancement of novel therapeutic strategies to treat infection. Mesenchymal stromal cells (MSC) possess antimicrobial and immunomodulatory properties, mediated through antimicrobial peptide secretion and recruitment of innate immune cells including neutrophils and monocytes. TLR-3 activation of human, canine and equine MSC has been shown to enhance bacterial killing and clearance in vitro, in rodent Staphylococcal biofilm infection models and dogs with spontaneous multi-drug-resistant infections. The objective of this study was to determine if intra-articular (IA) TLR-3-activated MSC with antibiotics improved clinical parameters and reduced bacterial counts and inflammatory cytokine concentrations in synovial fluid (SF) of horses with induced septic arthritis. Methods: Eight horses were inoculated in one tarsocrural joint with multidrug-resistant Staphylococcus aureus (S. aureus). Bone marrow-derived MSC from three unrelated donors were activated with TLR-3 agonist polyinosinic, polycytidylic acid (pIC). Recipient horses received MSC plus vancomycin (TLR-MSC-VAN), or vancomycin (VAN) alone, on days 1, 4, 7 post-inoculation and systemic gentamicin. Pain scores, quantitative bacterial counts (SF, synovium), SF analyses, complete blood counts, cytokine concentrations (SF, plasma), imaging changes (MRI, ultrasound, radiographs), macroscopic joint scores and histologic changes were assessed. Results were reported as mean ± SEM. Results: Pain scores (d7, P=0.01, 15.2±0.2 vs. 17.9±0.5), ultrasound (d7, P=0.03, 9.0±0.6 vs. 11.8±0.5), quantitative bacterial counts (SF d7, P=0.02, 0±0 vs. 3.4±0.4; synovium P=0.003, 0.4±0.4 vs. 162.7±18.4), systemic neutrophil (d4, P=0.03, 4.6±0.6 vs. 7.8±0.6) and serum amyloid A (SAA) (d4, P=0.01, 1,106.0±659.0 vs. 2,858.8±141.3; d7, P=0.02, 761.8±746.2 vs. 2,357.3±304.3), and SF lactate (d7, P<0.0001, 5.4±0.2 vs. 15.0±0.3), SAA (endterm, P=0.01, 0.0 vs. 2,094.0±601.6), IL-6 (P=0.03, 313.0±119.2 vs. 1,328.2±208.9), and IL-18 (P=0.02, 11.1±0.5 vs. 13.3±3.8) were improved in TLR-MSC-VAN vs. VAN horses. Study limitations include the small horse sample size, short study duration, and lack of additional control groups. Conclusions: Combined TLR-activated MSC with antibiotic therapy may be a promising approach to manage joint infections with drug resistant bacteria.

Canine polarized macrophages express distinct functional and transcriptomic profiles.

Macrophage differentiation and function in disease states is highly regulated by the local microenvironment. For example, macrophage exposure to IFN-γ (interferon gamma) initiates the development of inflammatory (M1) macrophages, which acquire anti-tumoral and antimicrobial activity, while exposure to IL-4 (interleukin-4) and IL-13 (interleukin-13) drives an anti-inflammatory (M2) macrophage phenotype, which promotes healing and suppression of inflammatory responses. Previous studies of canine polarized macrophages have identified several surface markers that distinguished GM-CSF (granulocyte macrophage colony stimulating factor), IFN-γ and LPS (lipopolysaccharide) derived M1 macrophages or M2 macrophages; and reported a subset of genes that can be used to differentiate between polarization states. However, the need remains to understand the underlying biological mechanisms governing canine macrophage polarization states. Therefore, in the present study we used transcriptome sequencing, a larger panel of flow cytometry markers, and the addition of antimicrobial functional assays to further characterize canine macrophage polarization. Transcriptome analysis revealed unique, previously unreported signatures and pathways for polarized canine M1 and M2 macrophages. New flow cytometric markers were also identified, along with new characterization of how macrophage polarization impacted antimicrobial functions. Taken together, the findings reported here provide new insights into canine macrophage biology and identify new tools for the evaluation of polarized macrophages in dogs.

Immune Activated Cellular Therapy for Drug Resistant Infections: Rationale, Mechanisms, and Implications for Veterinary Medicine.

Antimicrobial resistance and biofilm formation both present challenges to treatment of bacterial infections with conventional antibiotic therapy and serve as the impetus for development of improved therapeutic approaches. Mesenchymal stromal cell (MSC) therapy exerts an antimicrobial effect as demonstrated in multiple acute bacterial infection models. This effect can be enhanced by pre-conditioning the MSC with Toll or Nod-like receptor stimulation, termed activated cellular therapy (ACT). The purpose of this review is to summarize the current literature on mechanisms of antimicrobial activity of MSC with emphasis on enhanced effects through receptor agonism, and data supporting use of ACT in treatment of bacterial infections in veterinary species including dogs, cats, and horses with implications for further treatment applications. This review will advance the field's understanding of the use of activated antimicrobial cellular therapy to treat infection, including mechanisms of action and potential therapeutic applications.

In Vitro Anti-Inflammatory and Regenerative Effects of Autologous Conditioned Serum from Dogs with Osteoarthritis.

Osteoarthritis (OA) is mostly incurable and non-regenerative with long-term complications. Autologous conditioned serum (ACS), which is enriched in Interleukin 1 receptor antagonists (IL-1RA) and growth factors, could be an alternative treatment to accelerate the positive therapeutic effects. ACS is proposed to alleviate inflammation by blocking IL-1 receptors. However, to date, there is no report focusing on the cell-mediated anti-inflammation and regenerative effect caused by ACS, especially the ACS from patients. Therefore, this study aims to investigate the therapeutic potential of ACS generated from dogs with spontaneous OA, focusing on its promising anti-inflammatory and regenerative properties in vitro compared to the matched plasma. We found that ACS prepared from ten OA dogs contained significant concentrations of IL-1RA, vascular endothelial growth factor, and transforming growth factor beta, which are key cytokines in anti-inflammation and angiogenesis. Furthermore, we found that ACS suppressed T cell activity by reducing proliferation of effector T cells and simultaneously expanding numbers of immune suppressive FOXP3+ T cells. Lastly, we showed that ACS enhanced the proliferation of osteocytes and fibroblasts and promoted extracellular matrix gene expression in primary chondrocyte culture. Therefore, these studies indicate that ACS prepared from dogs with OA is active as an immunomodulatory and regenerative strategy for use in OA management.

Activated Mesenchymal Stromal Cell Therapy for Treatment of Multi-Drug Resistant Bacterial Infections in Dogs.

New and creative approaches are required to treat chronic infections caused by increasingly drug-resistant strains of bacteria. One strategy is the use of cellular therapy employing mesenchymal stromal cells (MSC) to kill bacteria directly and to also activate effective host immunity to infection. We demonstrated previously that activated MSC delivered systemically could be used effectively together with antibiotic therapy to clear chronic biofilm infections in rodent models. Therefore, we sought in the current studies to gain new insights into the antimicrobial properties of activated canine MSC and to evaluate their effectiveness as a novel cellular therapy for treatment of naturally-occurring drug resistant infections in dogs. These studies revealed that canine MSC produce and secrete antimicrobial peptides that synergize with most classes of common antibiotics to trigger rapid bactericidal activity. In addition, activated canine MSC migrated more efficiently to inflammatory stimuli, and secreted factors associated with wound healing and fibroblast proliferation and recruitment of activated neutrophils. Macrophages incubated with conditioned medium from activated MSC developed significantly enhanced bactericidal activity. Clinical studies in dogs with chronic multidrug resistant infections treated by repeated i.v. delivery of activated, allogeneic MSC demonstrated significant clinical benefit, including infection clearance and healing of infected tissues. Taken together, the results of these studies provide new insights into antimicrobial activity of canine MSC, and their potential clinical utility for management of chronic, drug-resistant infections.