Non-specific protection from respiratory tract infections in cattle generated by intranasal administration of an innate immune stimulant

Abstract

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 INFγ 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.

Fig 1. Treatment of PBMC with LTC upregulates secretion of IFNγ and MHCII expression in a dose-dependent manner.

Peripheral blood mononuclear cells from 5 different healthy animals were cultured in vitro and treated with increasing concentrations of LTC for 48 h, as noted in Methods. Supernatants were collected and analyzed for IFNγ secretion by ELISA (A). Untreated or LTC-treated PBMC were collected and stained for T cells (CD3⁺) or monocytes (CD14⁺) and CD14⁺ cells were analyzed for expression of MHCII (B). Similar results for MHCII expression were obtained for three separate experiments. Untreated or Concanavalin A (ConA) treated PBMC were used as negative and positive controls, respectively. Significant differences between either untreated animals or those either treated with ConA or LTC were determined using an ordinary one-way ANOVA with p values of ****, <0.0001 and **, <0.01.

Fig 2. Treatment of monocyte derived macrophages with LTC stimulates upregulation of MHCII expression and secretion of TNFα and IL-6.

Monocytes were prepared from PBMC from 3 different animals and differentiated for 7 days in vitro in 20 ng/ml human M-CSF. Monocyte-derived macrophages (MDM) were then treated for 36 h with LPS, INFγ, LTC, or LTC plus IFNγ at the indicated concentrations. MDM were analyzed for MHCII expression by flow cytometry and released cytokines were measured via ELISA. To normalize the absolute differences between animals, levels of secretion of TNFα and IL-6 were normalized to basal, unstimulated concentrations. Statistical analyses were performed using an ordinary one-way ANOVA with *, P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.001 and ‘ns’, not significant.

Fig 3. LTC treatment stimulates nitric oxide release by macrophages.

Monocyte derived macrophages were treated for 36 h with either LPS or LTC at the indicated concentrations. Culture supernatants were assays for NO indirectly by measuring levels of nitrite ion (NO₂^-) release into MDM culture supernatants using the Griess reagent for detection of nitrate release. Significant differences were determined using an ordinary one-way ANOVA with; ***, P<0.005.

Fig 4. Macrophage treatment with LTC triggers bactericidal activity.

Monocyte-derived macrophages from 3 animals were infected at a MOI = 5 for 3 h with S. aureus (A) M. hemolytica (B), or P. multocida (C), after which bactericidal activity was determined as noted in Methods. Each symbol represents one animal. Bactericidal activity was compared between untreated, LTC treated and aminoguanidine (AG) (10nM) pretreatment of LTC-treated macrophages. Comparisons between untreated, treated and AG pretreated cells for each bacterial pathogen were performed using an ordinary one-way ANOVA with *, P<0.05, ***, P<0.005.

Fig 5. Intranasal administration of LTC elicits cellular influx into the nasopharynx in a dose-dependent fashion in healthy cattle.

Four groups of healthy cattle (n = 5 per group) were treated intranasally with PBS (no treatment) or with 3 different dosages of LTC (1 ml per nostril (blue line), 2 ml per nostril (green line), or 3 ml per nostril (red line)). Nasopharyngeal swab samples were obtained at various intervals after treatment, and collected cells were analyzed by flow cytometry. Pharyngeal cells were also analyzed for increases in overall cellularity (A) and numbers of CD14⁺ monocytes (B) and expression of MHCII (C). Comparisons of differences in percentages of the number of live cells, CD14⁺ cells and mean fluorescence intensity (MFI) of MHCII between groups were evaluated as a function of time after treatment and dosage of LTC. Analysis of variance of these parameters was evaluated using a two-way ANOVA with, **P <0.010; and ***P<0.005.

Fig 6. Pharyngeal cells from LTC treated animals show increased expression of IFNγ, IL-8 and MCP-1 gene transcripts.

RNA was purified from nasopharyngeal swab samples obtained from cattle (n = 5 per group) treated with PBS or LTC (2 ml per nostril) at the indicated times post-treatment. cDNA was reverse transcribed and amplified by qRT-PCR. Transcript numbers were compared after 6h, 24h, 72h, 7days, and 2 weeks of treatment and plotted accordingly. Statistical differences in PBS-treated versus LTC-treated groups were evaluated using a two-way ANOVA with **P <0.010; and ***P<0.005.

Fig 7. Intranasal LTC treatment generates increased serum IgG binding to BRDC pathogenic bacteria.

Serum obtained from PBS treated or LTC treated (2 ml per nostril) cattle (n = 6 per groups) was incubated with BRDC bacteria in vitro, and IgG binding determined by flow cytometry, as described in Methods. Serum IgG binding to M. hemolytica (A), P. multocida (B) and H. somni (C) is depicted, comparing untreated versus LTC treated animals. Antibody binding intensity was displayed as geometric mean fluorescence intensity of IgG positive bacteria. Significant differences were detected using an unpaired t test with, *, P<0.05 and **, P<0.01.

Fig 8. Impact of intranasal immunotherapy on the nasal microbiome.

Bacteria were recovered from nasal swabs obtained from cattle (n = 10 per group) treated by intranasal administration of PBS or LTC (2 ml per nostril). Swabs were obtained pre-treatment and again at 7 days after treatment, bacteria were isolated and DNA extracted, and subjected to 16S sequencing at a commercial laboratory, as described in Methods. Analysis of sequence information revealed the following: (A) Diversity analysis of Simpson’s evenness measure depicted on x-axis, y-axis represents phylogenic diversity (PD). Red bars represent pretreatment diversity in the LTC-treated group, orange represents 7 days after treatment in the LTC-treated group. Blue and green bars represent pre and 7 day valued in the control (PBS-treated) group. (1B) Faith alpha diversity. (1C) Principal components analysis of Bray-Curtis distance beta diversity (color legend in top right corner). Proportion of variance explained by each principal coordinate axis is denoted in the corresponding axis label. (1D) principal component analysis of Jaccard beta diversity.

Fig 9. Impact of LTC pre-treatment on clinical illness scores in cattle subjected to BRDC challenge.

Cattle (n = 24 per group) were treated with PBS (2 ml per nostril) or LTC (2 ml per nostril), and then 24 h later were co-mingled with BHV-1 infected, BVD infected, and M. haemolytica- infected seeder animals, as described in Methods. All exposed treated animals were monitored daily for clinical illness scores by blinded clinical observers, and overall scores tallied for each group of animals for the entire 24-day study period and mean and SEM values for illness scores were plotted. Data were analyzed for significance using a 2-way ANOVA with **, P<0.01.

Fig 10. LTC pre-treatment significantly reduces euthanasia (mortality) associated with experimental BRDC infection in cattle.

Cattle (n = 24 per group) were treated with PBS (2 ml per nostril) or LTC (2 ml per nostril), and then 24h later were co-mingled with BHV-1 infected, BVD infected, and M. haemolytica-infected seeder animals, as described in Methods. Animals in each treatment group were monitored daily for signs of clinical illness by observers and were euthanized when clinical scores reached a pre-determined score. Survival was evaluated and represented by Kaplan-Meier survival curve, followed by log-rank analysis to determine the level of statistical significance (p = 0.02) (A). In animals pre-treated with LTC, the total percentage of animals euthanized due to BRDC associated clinical illness was 12.5%, which was markedly lower than the 42% euthanized due to clinical illness for animals in the PBS treated group (B).