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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.

Mesenchymal stem cells (MSC) have been shown to improve wound healing and suppress inflammatory immune responses. Newer research also indicates that MSC exhibit antimicrobial activity, although the mechanisms underlying this activity have not been fully elucidated. Therefore, we conducted in vitro and in vivo studies to examine the ability of resting and activated MSC to kill bacteria, including multidrug resistant strains. We investigated direct bacterial killing mechanisms and the interaction of MSC with host innate immune responses to infection. In addition, the activity of MSC against chronic bacterial infections was investigated in a mouse biofilm infection model. We found that MSC exhibited high levels of spontaneous direct bactericidal activity in vitro. Moreover, soluble factors secreted by MSC inhibited Staphylococcus aureus biofilm formation in vitro and disrupted the growth of established biofilms. Secreted factors from MSC also elicited synergistic killing of drug‐resistant bacteria when combined with several major classes of antibiotics. Other studies demonstrated interactions of activated MSC with host innate immune responses, including triggering of neutrophil extracellular trap formation and increased phagocytosis of bacteria. Finally, activated MSC administered systemically to mice with established S. aureus biofilm infections significantly reduced bacterial numbers at the wound site and improved wound healing when combined with antibiotic therapy. These results indicate that MSC generate multiple direct and indirect, immunologically mediated antimicrobial activities that combine to help eliminate chronic bacterial infections when the cells are administered therapeutically. Human bone marrow MSC secrete factors that interact synergistically with antibiotics to media direct bacterial killing. Activation of MSC enhances indirect mechanisms of bacterial killing in a mouse biofilm infection model, and facilitates increased anti bacterial activity of neutrophils.

Cellular therapy with allogeneic or autologous mesenchymal stem cells (MSC) has emerged as a promising new therapeutic strategy for managing inflammatory bowel disease (IBD). However, MSC therapy ideally requires a convenient and relatively homogenous cell source (typically bone marrow or adipose tissues) and the ability to generate cells with stable phenotype and function. An alternative means of generating allogeneic MSC is to derive them from induced pluripotent stem cells (iPSC), which could in theory provide an indefinite supply of MSC with well‐defined phenotype and function. Therefore, we compared the effectiveness of iPSC‐derived MSC (iMSC) and adipose‐derived MSC (adMSC) in a mouse model of IBD (dextran sodium sulfate‐induced colitis), and investigated mechanisms of intestinal protection. We found that iMSC were equivalent to adMSC in terms of significantly improving clinical abnormalities in treated mice and reducing lesion scores and inflammation in the gut. Administration of iMSC also stimulated significant intestinal epithelial cell proliferation, increased in the numbers of Lgr5+ intestinal stem cells, and increased intestinal angiogenesis. In addition, the microbiome alterations present in mice with colitis were partially restored to resemble those of healthy mice following treatment with iMSC or adMSC. Thus, iMSC administration improved overall intestinal health and healing with equivalent potency to treatment with adMSC. This therefore is the first report of the effectiveness of iMSC in the treatment of IBD, along with a description of unique mechanisms of action with respect to intestinal healing and microbiome restoration. Stem Cells Translational Medicine 2018;7:456–467 Graphical summarizing the effects of stem cell treatment on the colonic tissue, intestinal stem cells, and fecal microbiome

Immune cells play key roles in host responses to malignant tumors. The selective pressure that immune cells elicit on tumors promotes immune escape, while tumor-associated modulation of immune cells creates an environment favorable to tumor growth and progression. In this study we used publicly available single-cell RNA sequencing (scRNA-seq) data from the translationally relevant canine osteosarcoma (OS) model to compare tumor-infiltrating immune cells to circulating leukocytes. Through computational analysis we investigated the differences in cell type proportions and how the OS TME impacted infiltrating immune cell transcriptomic profiles relative to circulating leukocytes. Differential abundance analysis revealed increased proportions of follicular helper T cells, regulatory T cells, and mature regulatory dendritic cells (mregDCs) in the OS TME. Differential gene expression analysis identified exhaustion markers (LAG3, HAVCR2, PDCD1) to be upregulated in CD4 and CD8 T cells within the OS TME. Comparisons of B cell gene expression profiles revealed an enrichment of protein processing and endoplasmic reticulum pathways, suggesting infiltrating B cells were activated following tumor infiltration. Gene expression changes within myeloid cells identified increased expression of immune suppressive molecules (CD274, OSM, MSR1) in the OS TME, indicating the TME skews myeloid cells toward an immunosuppressive phenotype. Comparisons to human literature and analysis of human scRNA-seq data revealed conserved transcriptomic responses to tumor infiltration, while also identifying species differences. Overall, the analysis presented here provides new insights into how the OS TME impacts the transcriptional programs of major immune cell populations in dogs and acts as a resource for comparative immuno-oncology research.

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.

Inflammatory bowel disease (IBD) in dogs is associated with clinical signs of intestinal dysfunction, as well as abnormal lymphocytic and myeloid cell infiltrates in the small and/or large intestine. Thus, in many respects IBD in dogs resembles IBD in humans. However, the factors that trigger intestinal inflammation in dogs with IBD are not well understood and have been variously attributed to immune responses against dietary antigens or intestinal antigens. Previous studies in humans with IBD have documented increased production of IgG and IgA antibodies specific to intestinal bacteria, and this abnormal immune response has been linked to disease pathogenesis. Therefore, we investigated the humoral immune response against gut bacteria in dogs with IBD, using flow cytometry to quantitate IgG and IgA binding. Studies were also done to investigate the source of these antibodies (locally produced versus systemic production) and whether greater antibody binding to bacteria is associated with increased inflammatory responses. We found that dogs with IBD had significantly higher percentages and overall amounts of IgG bound to their intestinal bacteria compared to healthy dogs. Similarly, significantly higher percentages of bacteria were IgA+ bacteria were also found in dogs with IBD. Serum antibody recognition of gut bacteria was not different between healthy dogs and dogs with IBD, suggesting that anti-bacterial antibodies were primarily produced locally in the gut rather than systemically. Importantly, bacteria in the Actinobacteria phylum and in particular the genus Collinsella had significantly greater levels of antibody binding in dogs with IBD. Based on these findings, we concluded that antibody binding to commensal gut bacteria was significantly increased in dogs with IBD, that particular phyla were preferential targets for gut antibodies, and that anti-bacterial antibody responses may play an important role in regulating gut inflammation.

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.

Alternatives to antibiotics for prevention of respiratory tract infections in cattle are urgently needed given the increasing public and regulatory pressure to reduce overall antibiotic usage. Activation of local innate immune defenses in the upper respiratory tract is one strategy to induce non-specific protection against infection with the diverse array of viral and bacterial pathogens associated with bovine respiratory disease complex (BRDC), while avoiding the use of antibiotics. Our prior studies in rodent models demonstrated that intranasal administration of liposome-TLR complexes (LTC) as a non-specific immune stimulant generated high levels of protection against lethal bacterial and viral pathogens. Therefore, we conducted studies to assess LTC induction of local immune responses and protective immunity to BRDC in cattle. In vitro, LTC were shown to activate peripheral blood mononuclear cells in cattle, which was associated with secretion of INF[gamma] and IL-6. Macrophage activation with LTC triggered intracellular killing of Mannheimia hemolytica and several other bacterial pathogens. In studies in cattle, intranasal administration of LTC demonstrated dose-dependent activation of local innate immune responses in the nasopharynx, including recruitment of monocytes and prolonged upregulation (at least 2 weeks) of innate immune cytokine gene expression by nasopharyngeal mucosal cells. In a BRDC challenge study, intranasal administration of LTC prior to pathogen exposure resulted in significant reduction in both clinical signs of infection and disease-associated euthanasia rates. These findings indicate that intranasal administration of a non-specific innate immune stimulant can be an effective method of rapidly generating generalized protection from mixed viral and bacterial respiratory tract infections in cattle.

To determine the safety and efficacy of perilesional human recombinant interferon alpha-2b (IFNα2b) for treatment of periocular squamous cell carcinoma (PSCC) in horses. Eleven horses (12 eyes) with PSCC were enrolled in this prospective clinical study with owner consent. 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. 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. 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.

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.