Effect of aerosolized bacterial lysate on development of naturally occurring respiratory disease in beef calves.

BACKGROUND: Bovine respiratory disease (BRD) is a major problem affecting beef cattle after arrival to feedlots. Alternatives to antibiotics are needed for prevention. HYPOTHESIS: Stimulation of pulmonary innate immune responses at the time of arrival to a feedlot reduces the occurrence and severity of BRD. ANIMALS: Sixty beef steers at high risk of BRD. METHODS: Randomized, double-blinded, placebo-controlled study. Calves received saline or a lysate of Staphylococcus aureus and Escherichia coli by aerosol, at 16 hours after feedlot arrival. Calves were monitored for 28 days for disease outcomes and levels of Mycoplasma bovis and Mannheimia haemolytica in nasal swabs. RESULTS: Death from M bovis pneumonia was significantly greater in lysate-treated animals (6/29, 24%) compared to controls (1/29, 3%; odds ratio = 10.2; 95% confidence interval [CI] = 1.1-96.0; P = .04). By 28 days after arrival, 29/29 lysate-treated calves had ultrasonographic pulmonary consolidation compared to 24/29 control calves (P = .05). Lysate-treated calves had lower weight gain compared to control calves (-8.8 kg, 95% CI = -17.1 to -0.5; P = .04), and higher body temperatures on days 4, 7, and 21 (0.19°C; 95% CI = 0.01-0.37; P = .04). Nasal M bovis numbers increased over time and were higher in lysate-treated calves (0.76 log CFU, 95% CI = 0.3-1.2; P = .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Aerosol administration of a bacterial lysate exacerbated BRD in healthy high-risk beef calves, suggesting that respiratory tract inflammation adversely affects how calves respond to subsequent natural infection with M bovis and other respiratory pathogens.

Pulmonary and systemic responses to aerosolized lysate of Staphylococcus aureus and Escherichia coli in calves.

BACKGROUND: Constitutive and inducible defenses protect the respiratory tract from bacterial infection. The objective of this study was to characterize the response to an aerosolized lysate of killed bacteria, as a basis for studying the regulation and in vivo effects of these inducible innate immune responses. RESULTS: Bacterial lysate consisting of heat-killed and sonicated Staphylococcus aureus and Escherichia coli was aerosolized to 6 calves and systemic and pulmonary innate immune and inflammatory responses were measured in the first 24 h relative to baseline. Evaluated parameters included clinical parameters (body temperature and heart and respiratory rates), blood acute phase proteins and leukocyte counts, and leukocytes and proteins in bronchoalveolar lavage fluid. Mild clinical signs with increased heart rates and rectal temperatures developed following administration of the lysate, with resolution by 24 h. Serum haptoglobin and plasma fibrinogen concentrations were elevated at 24 h relative to baseline. Bronchoalveolar lavage fluid (BALF) had increased cellularity and increased proportion of neutrophils, as well as higher concentrations of interleukin (IL)-8, IL-10 and total protein at 24 h relative to baseline. Mass spectrometry identified 965 unique proteins in BALF: 19 proteins were increased and 26 proteins were decreased relative to baseline. The upregulated proteins included those involved in innate immunity including activation of complement, neutrophils and platelets. At postmortem examination, calves receiving higher doses of lysate had areas of lobular consolidation and interlobular edema. Histologically, neutrophils were present within bronchioles and to a lesser extent within alveoli. Calves receiving highest doses of lysate had patchy areas of neutrophils, hemorrhage and hyaline membranes within alveoli. CONCLUSIONS: Aerosolization of bacterial lysate stimulated an innate immune response in lungs and airways, with alveolar damage observed at higher doses. Such a stimulus could be of value for investigating the effects of inducible innate immune responses on occurrence of disease, or for evaluating how stress, drugs or genetics affect these dynamic responses of the respiratory tract.

Safety of an extended-release injectable moxidectin suspension formulation (ProHeart® 12) in dogs.

BACKGROUND: The safety of ProHeart® 12 (PH 12; extended-release injectable suspension; 10% moxidectin in glyceryl tristearate microspheres) was evaluated in four studies using Beagle dogs and one study using ivermectin-sensitive Collies. The recommended dose is 0.5 mg/kg subcutaneously once yearly. METHODS: Study 1: safety margin was evaluated as 3 treatments of PH 12 (0× (control); 1× (recommended dose); 3× (3 times recommended dose) and 5× (5 times recommended dose) in 12 months via clinical observations, body weights, food consumption, injection site observations, physical examinations, moxidectin tissue assay, pharmacokinetics, and clinical and anatomic pathology. Study 2: safety in breeding-age males was demonstrated by semen testing at 14-day intervals from Day 7 to Day 91 post-treatment (0× or 3×). Study 3: reproductive safety in females was demonstrated by monitoring dams and litters following treatments (0× or 3×) administered during breeding, gestation, or lactation. Study 4: safety in dogs surgically implanted with adult heartworms was evaluated by clinical and laboratory monitoring following treatment with 0× or 3× administered 61 days post-implantation. Study 5: safety in ivermectin-sensitive dogs (120 µg/kg SC) was by clinical monitoring for 1 week after administering 1×, 3× or 5×. RESULTS: Study 1: slight swelling clinically detectable at some 3× and 5× injection sites was characterized microscopically as granulomatous inflammation, like tissue responses to medical implants, interpreted as non-adverse. Pharmacokinetics were dose-proportional and there was little or no systemic accumulation. Residual moxidectin mean (range) at 1× injection sites after 1 year was 16.0% (0.045-37.6%) of the administered mass. Studies 2 and 3: no effects were identified in reproductive indices (females) or semen quality characteristics (males). Study 4: PH 12 produced marked reductions in circulating microfilariae and lower numbers of adult heartworms, but no adverse clinical signs were identified. Study 5: there were no abnormal clinical signs at 1×, 3× or 5× overdoses of PH 12 in ivermectin-sensitive dogs. CONCLUSIONS: PH 12 has a > 5× safety margin in both normal and ivermectin-sensitive dogs, has no effects on canine reproduction, and is well tolerated in heartworm-positive dogs. The only treatment-related finding was non-adverse, granulomatous inflammation at the injection site.

Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity.

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune, skin, and neuronal target cells.

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.

Comparative functional characterization of canine IgG subclasses.

To date, very little is known about the functional characteristics of the four published canine IgG subclasses. It is not clear how each subclass engages the immune system via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), or how long each antibody may last in serum. Such information is critical for understanding canine immunology and for the discovery of canine therapeutic monoclonal antibodies. Through both in vitro and ex vivo experiments to evaluate canine Fc's for effector function, complement binding, FcRn binding, and ADCC, we are now able to categorize canine subclasses by function. The subclasses share functional properties with the four human IgG subclasses and are reported herein with their function-based human analog. Canine Fc fusions, canine chimeras, and caninized antibodies were characterized. Canine subclasses A and D appear effector-function negative while subclasses B and C bind canine Fc gamma receptors and are positive for ADCC. All canine subclasses bind the neonatal Fc receptor except subclass C. By understanding canine IgGs in this way, we can apply what is known of human immunology toward translational and veterinary medicine. Thus, this body of work lays the foundation for evaluating canine IgG subclasses for therapeutic antibody development and builds upon the fundamental scholarship of canine immunology.

Hydrocortisone inhibits IFN-γ production in equine, ovine, and bovine PBMCs.

Hydrocortisone is widely accepted as an anti-inflammatory agent and there are many products available containing hydrocortisone as an active ingredient. Surprisingly, there is little data available specifically on the immunological effects of hydrocortisone in large animals. Glucocorticoids are well-characterized for their ability to repress inflammation via a wide variety of mechanisms including suppression of cytokine production. In this study the effects of hydrocortisone on IFN-γ production by equine, bovine, and ovine PBMCs were assessed using flow cytometric or ELISpot analysis. Hydrocortisone suppressed mitogen-driven IFN-γ production by PBMCs from all three species of animals, confirming that this agent mediates anti-inflammatory effects in large animals. Although the results from this study were expected based on the precedence set in murine and human systems, it is important to understand the effects of administration of a compound or product in the species of interest as species-specific indications are not always available.

CXCR7 influences leukocyte entry into the CNS parenchyma by controlling abluminal CXCL12 abundance during autoimmunity.

Loss of CXCL12, a leukocyte localizing cue, from abluminal surfaces of the blood-brain barrier occurs in multiple sclerosis (MS) lesions. However, the mechanisms and consequences of reduced abluminal CXCL12 abundance remain unclear. Here, we show that activation of CXCR7, which scavenges CXCL12, is essential for leukocyte entry via endothelial barriers into the central nervous system (CNS) parenchyma during experimental autoimmune encephalomyelitis (EAE), a model for MS. CXCR7 expression on endothelial barriers increased during EAE at sites of inflammatory infiltration. Treatment with a CXCR7 antagonist ameliorated EAE, reduced leukocyte infiltration into the CNS parenchyma and parenchymal VCAM-1 expression, and increased abluminal levels of CXCL12. Interleukin 17 and interleukin 1ß increased, whereas interferon-γ decreased, CXCR7 expression on and CXCL12 internalization in primary brain endothelial cells in vitro. These findings identify molecular requirements for the transvascular entry of leukocytes into the CNS and suggest that CXCR7 blockade may have therapeutic utility for the treatment of MS.

CXCR4 promotes differentiation of oligodendrocyte progenitors and remyelination.

Multiple sclerosis is a neurodegenerative disease characterized by episodes of autoimmune attack of oligodendrocytes leading to demyelination and progressive functional deficits. Because many patients exhibit functional recovery in between demyelinating episodes, understanding mechanisms responsible for repair of damaged myelin is critical for developing therapies that promote remyelination and prevent disease progression. The chemokine CXCL12 is a developmental molecule known to orchestrate the migration, proliferation, and differentiation of neuronal precursor cells within the developing CNS. Although studies suggest a role for CXCL12 in oligodendroglia ontogeny in vitro, no studies have investigated the role of CXCL12 in remyelination in vivo in the adult CNS. Using an experimental murine model of demyelination mediated by the copper chelator cuprizone, we evaluated the expression of CXCL12 and its receptor, CXCR4, within the demyelinating and remyelinating corpus callosum (CC). CXCL12 was significantly up-regulated within activated astrocytes and microglia in the CC during demyelination, as were numbers of CXCR4+NG2+ oligodendrocyte precursor cells (OPCs). Loss of CXCR4 signaling via either pharmacological blockade or in vivo RNA silencing led to decreased OPCs maturation and failure to remyelinate. These data indicate that CXCR4 activation, by promoting the differentiation of OPCs into oligodendrocytes, is critical for remyelination of the injured adult CNS.

IL-1R signaling within the central nervous system regulates CXCL12 expression at the blood-brain barrier and disease severity during experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the CNS characterized by disruption of the blood-brain barrier (BBB). This breach in CNS immune privilege allows undeterred trafficking of myelin-specific lymphocytes into the CNS where they induce demyelination. Although the mechanism of BBB compromise is not known, the chemokine CXCL12 has been implicated as a molecular component of the BBB whose pattern of expression is specifically altered during MS and which correlates with disease severity. The inflammatory cytokine IL-1beta has recently been shown to contribute not only to BBB permeability but also to the development of IL-17-driven autoimmune responses. Using experimental autoimmune encephalomyelitis, the rodent model of MS, we demonstrate that IL-1beta mediates pathologic relocation of CXCL12 during the induction phase of the disease, before the development of BBB disruption. We also show that CD4, CD8, and, surprisingly gammadelta T cells are all sources of IL-1beta. In addition, gammadelta T cells are also targets of this cytokine, contributing to IL-1beta-mediated production of IL-17. Finally, we show that the level of CNS IL-1R determines the clinical severity of experimental autoimmune encephalomyelitis. These data suggest that T cell-derived IL-1beta contributes to loss of immune privilege during CNS autoimmunity via pathologic alteration in the expression of CXCL12 at the BBB.

CXCR4 antagonism increases T cell trafficking in the central nervous system and improves survival from West Nile virus encephalitis.

The migration of lymphocytes into the CNS during viral encephalitis is hindered by the blood-brain barrier (BBB) such that most infiltrating cells remain localized to perivascular spaces. This sequestration of leukocytes away from the parenchyma is believed to protect the CNS from immunopathologic injury. Infections of the CNS with highly cytopathic neurotropic viruses, such as West Nile virus (WNV), however, require the parenchymal penetration of T lymphocytes for virus clearance and survival, suggesting that perivascular localization might hinder antiviral immune responses during WNV encephalitis. Using human and murine brain specimens from individuals with WNV encephalitis, we evaluated the expression of CXCL12 and its receptor, CXCR4, at the BBB and tested the hypothesis that inhibition of CXCR4 would promote T lymphocyte entry into the CNS parenchyma and increase viral clearance. Antagonism of CXCR4 significantly improved survival from lethal infection through enhanced intraparenchymal migration of WNV-specific CD8(+) T cells within the brain, leading to reduced viral loads and, surprisingly, decreased immunopathology at this site. The benefits of enhanced CD8(+) T cell infiltration suggest that pharmacologic targeting of CXCR4 may have therapeutic utility for the treatment of acute viral infections of the CNS.

Pathological expression of CXCL12 at the blood-brain barrier correlates with severity of multiple sclerosis.

Dysregulation of blood-brain barrier (BBB) function and transendothelial migration of leukocytes are essential components of the development and propagation of active lesions in multiple sclerosis (MS). Animal studies indicate that polarized expression of the chemokine CXCL12 at the BBB prevents leukocyte extravasation into the central nervous system (CNS) and that disruption of CXCL12 polarity promotes entry of autoreactive leukocytes and inflammation. In the present study, we examined expression of CXCL12 and its receptor, CXCR4, within CNS tissues from MS and non-MS patients. Immunohistochemical analysis of CXCL12 expression at the BBB revealed basolateral localization in tissues derived from non-MS patients and at uninvolved sites in tissues from MS patients. In contrast, within active MS lesions, CXCL12 expression was redistributed toward vessel lumena and was associated with CXCR4 activation in infiltrating leukocytes, as revealed by phospho-CXCR4-specific antibodies. Quantitative assessment of CXCL12 expression by the CNS microvasculature established a positive correlation between CXCL12 redistribution, leukocyte infiltration, and severity of histological disease. These results suggest that CXCL12 normally functions to localize infiltrating leukocytes to perivascular spaces, preventing CNS parenchymal infiltration. In the patient cohort studied, altered patterns of CXCL12 expression at the BBB were specifically associated with MS, possibly facilitating trafficking of CXCR4-expressing mononuclear cells into and out of the perivascular space and leading to progression of disease.

CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis.

Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40(-/-) mice uniformly succumbed to WNV infection. Although CD40(-/-) mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40(-/-) mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40(-/-) mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.

Molecular targets for disrupting leukocyte trafficking during multiple sclerosis.

Autoimmune diseases of the central nervous system (CNS) involve the migration of abnormal numbers of self-directed leukocytes across the blood-brain barrier that normally separates the CNS from the immune system. The cardinal lesion associated with neuroinflammatory diseases is the perivascular infiltrate, which comprises leukocytes that have traversed the endothelium and have congregated in a subendothelial space between the endothelial-cell basement membrane and the glial limitans. The exit of mononuclear cells from this space can be beneficial, as when virus-specific lymphocytes enter the CNS for pathogen clearance, or might induce CNS damage, such as in the autoimmune disease multiple sclerosis when myelin-specific lymphocytes invade and induce demyelinating lesions. The molecular mechanisms involved in the movement of lymphocytes through these compartments involve multiple signalling pathways between these cells and the microvasculature. In this review, we discuss adhesion, costimulatory, cytokine, chemokine and signalling molecules involved in the dialogue between lymphocytes and endothelial cells that leads to inflammatory infiltrates within the CNS, and the targeting of these molecules as therapies for the treatment of multiple sclerosis.

CXCL12 limits inflammation by localizing mononuclear infiltrates to the perivascular space during experimental autoimmune encephalomyelitis.

The inflammatory response in the CNS begins with the movement of leukocytes across the blood-brain barrier in a multistep process that requires cells to pass through a perivascular space before entering the parenchyma. The molecular mechanisms that orchestrate this movement are not known. The chemokine CXCL12 is highly expressed throughout the CNS by microendothelial cells under normal conditions, suggesting it might play a role maintaining the blood-brain barrier. We tested this hypothesis in the setting of experimental autoimmune encephalomyelitis (EAE) by using AMD3100, a specific antagonist of the CXCL12 receptor CXCR4. We demonstrate that the loss of CXCR4 activation enhances the migration of infiltrating leukocytes into the CNS parenchyma. CXCL12 is expressed at the basolateral surface of CNS endothelial cells in normal spinal cord and at the onset of EAE. This polarity is lost in vessels associated with an extensive parenchymal invasion of mononuclear cells during the peak of disease. Inhibition of CXCR4 activation during the induction of EAE leads to loss of the typical intense perivascular cuffs, which are replaced with widespread white matter infiltration of mononuclear cells, worsening the clinical severity of the disease and increasing inflammation. Taken together, these data suggest a novel anti-inflammatory role for CXCL12 during EAE in that it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thereby limiting the parenchymal infiltration of autoreactive effector cells.

Region-specific regulation of inflammation and pathogenesis in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis and is characterized by an infiltrate of predominantly T cells and macrophages in the spinal cord and brain. In both the spinal cord and the cerebellum, Th1 cells direct inflammation to antigen-rich white matter tracts, and there is a TNFR1-dependent recruitment of CD11b(hi) cells in both regions. In the spinal cord, parenchymal invasion, demyelination and clinical symptoms are associated with TNFR1-dependant parenchymal induction (especially astrocytes) of VCAM-1 and CXCL2. None of these events occur in the cerebellum despite the fact that an inflammatory infiltrate accumulates in the perivascular space. Therefore regional specificity in astrocyte responses to inflammatory cytokines may regulate regional parenchymal infiltration and pathogenesis.