Inflammatory Profiles Induced by Intranasal Immunization with Ricin Toxin-immune Complexes Open Access

Inhalation of the biothreat agent ricin toxin (RT) provokes a localized inflammatory response that involves pulmonary congestion, edema, neutrophil infiltration, and severe acute respiratory distress (1). RT’s binding subunit (RTB), a galactose/N-acetyl galactosamine (Gal/GalNAc)-specific lectin, mediates toxin uptake into airway epithelial cells and other cell types within the mucosa (2, 3). Within the airway, RT is internalized by alveolar macrophages via mannose receptor–mediated recognition of RTB’s two-mannose side chain (4). RT’s enzymatic subunit (RTA) is a ribosome-inactivating protein that triggers cellular stress and programmed cell death pathways, resulting in the release of high levels of proinflammatory cytokines and chemokines (5–7).

Although there are currently no Food and Drug Administration–approved medical countermeasures for pulmonary RT exposure, early intervention with the toxin-neutralizing mAb PB10, which targets an immunodominant epitope on RTA, rescued Rhesus macaques from an otherwise lethal dose of aerosolized toxin (8). The same mAb given to nonhuman primates in advance of RT exposure conferred near-complete protection against toxin-induced morbidity and mortality (9). The efficacy of PB10 is further improved when combined with a second toxin-neutralizing mAb, SylH3, directed against RTB (10–12). Interestingly, although the PB10/SylH3 mixture virtually eliminates RT-induced pulmonary toxicity and tissue damage, the Ab mixture only marginally reduces RT binding to target cells in the lung tissues (11). Therefore, we have postulated that PB10 and SylH3 neutralize RT by interfering with RTB- and MR-mediated uptake and/or intracellular trafficking rather than blocking attachment to target cells (11, 13–15).

We were intrigued by the possibility that therapeutic Abs such as PB10 and SylH3 might influence the immunological fate of RT, because immune complexes (ICs) are known to have immunomodulatory activity (16). Indeed, we found that intranasal (i.n.) administration of ricin immune complexes (RICs) consisting of RT and equimolar amounts of PB10 and SylH3 resulted in the rapid onset of anti-RT IgG Abs that persisted for months (17). Moreover, a single i.n. dose of RIC was sufficient to confer protection against RT challenge 30 or 90 d later. The induction of RT-specific serum IgG was independent of FcγR engagement, as revealed through FcγR knockout mice and PB10/SylH3 LALA (leucine to alanine) derivatives. These observations suggested that RT as a preformed IC has the potential to generate a humoral response akin to vaccination. Understanding this phenomenon could potentially open up new avenues for both RT vaccine development and the expansion of therapeutic mAb applications for preventive purposes. In the present study, we set out to define the role of Th cells and characterize the local and systemic inflammatory environments in the context of i.n. RICs immunization.

RT (Ricinus communis agglutinin II; RCA₆₀) was custom ordered from Vector Laboratories (Burlingame, CA) and dialyzed against PBS at 4°C in 10,000 MWCO Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, Pittsburgh, PA) prior to use in mice. Murine mAbs PB10 and SylH3 were purified by protein G chromatography by the Dana-Farber Cancer Institute’s Monoclonal Antibody Core Facility. PB10 is a murine IgG2b that targets RTA and SylH3 an IgG1 targeting RTB (18–21). Unless noted otherwise, all reagents were purchased from Sigma-Aldrich (St. Louis, MO).

The mouse studies were conducted under strict compliance with the Wadsworth Center’s Institutional Animal Care and Use Committee. Female BALB/c mice aged 7–8 wk were purchased from Taconic Biosciences (Rensselaer, NY). For RIC immunizations, RT (1 μg) was mixed with 20 μg each of PB10 IgG2b and SylH3 IgG1 and administered to mice in a final volume of 40 μl i.n. For RT-only exposures, 5 × LD₅₀ (1 μg unless otherwise noted) was administered to mice in a volume of 40 μl for i.n. delivery. All i.n. inoculations were performed on isoflurane-anesthetized animals, which were monitored after the procedure to ensure normal recovery from anesthesia. Following RT exposure, mice were monitored daily for 7 d for symptoms of ricin intoxication, including weight loss, low blood glucose levels, and clinical signs of morbidity. Morbidity was measured using an institutional animal care and use committee–approved grading sheet stratified on a scale from 0 to 3, with 0 indicating normal activity and appearance and 3 indicating severe illness.

Mice were euthanized by CO₂ asphyxiation followed by cervical dislocation when they exceeded predetermined thresholds for weight loss, blood glucose levels, or physical signs of morbidity. For studies requiring harvest of bronchoalveolar lavage fluid (BALF), mice were euthanized by CO₂ asphyxiation and subsequent exsanguination via the abdominal aorta. For collection of BALF, 1 ml 0.6 mM EDTA in PBS was injected into the lungs intratracheally and immediately recollected. BALF samples were stored at −80°C until use. Blood was procured from the submandibular vein and serum isolated as described (17).

Mice were i.p. injected with 200 μg of rat anti-mouse CD4 mAb (clone GK1.5; Thermo Fisher Scientific) 1 d prior to RIC immunization. The same dose and delivery route were used for subsequent treatment on days 1 and 4 following RIC immunization. For confirmation of Ab efficacy, mice were i.p. injected with 200 μg rat anti-mouse CD4 mAb 1 d before harvest of splenocytes for analysis of CD4⁺ populations by flow cytometry in comparison with splenocytes from untreated animals. The following Abs were used for labeling cells for flow cytometry: PE rat anti-mouse CD45 (clone 30-F11; BioLegend), FITC rat anti-mouse CD3 (clone 17A2; BD Biosciences, San Jose, CA), and allophycocyanin rat anti-mouse CD4 (clone RM4-5; BD Biosciences).

Mice were i.p. injected with 400 μg InVivoMAb anti-mouse IL-6 mAb (clone MP5-20F3; Bio X Cell, Lebanon, NH) 1 d prior to RIC immunization. The same dose and delivery route were used for treatment 1 d after RIC immunization. For confirmation of Ab efficacy, mice were i.p. injected with 400 μg InVivoMAb anti-mouse IL-6 mAb 1 d before RIC immunization. On the day following immunization, BALF was collected for analysis of secreted IL-6 by Luminex in comparison with BALF from immunized animals that did not receive anti-mouse IL-6 treatment.

Ninety-six-well high-binding microtiter plates (Thermo Fisher Scientific) were coated overnight with RT (0.1 μg/well in PBS) at 4°C. The plates were then washed and blocked as described (17) before the addition of mouse sera or mAbs. Sera and mAbs were diluted in ELISA block solution (5% goat serum, 0.1% Tween 20 in PBS). HRP-labeled goat anti-mouse IgG polyclonal Abs (SouthernBiotech, Birmingham, AL) were diluted 1:2000 in block solution prior to use. Plates were developed using 3,30,5,50-tetramethylbenzidine (Kirkegaard & Perry Labs, Gaithersburg, MD) and analyzed with a SpectraMax iD3 spectrophotometer equipped with SoftMax Pro 7.1 software (Molecular Devices, Sunnyvale, CA).

The MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Panel (MCYTMAG-70K-PX32) from MilliporeSigma (Burlington, MA) was used to quantify cytokine contents from serum and BALF samples according to the manufacturer’s instructions. Serum and BALF samples were diluted in an equal volume of assay buffer and PBS, respectively, then added to premixed beads in a 96-well microtiter plate for overnight incubation at 4°C with shaking. Plates were washed using a handheld plate magnet before addition of primary Abs followed by incubation at room temperature for 1 h with shaking. Streptavidin-PE was added to each well and incubated with shaking for 30 min at room temperature. Plates were washed as described above and run in Sheath Fluid PLUS on a Luminex FLEXMAP 3D with INTELLIFLEX software (Luminex Corp., Austin, TX). Values were recorded as median fluorescence intensity. For analysis, assay kit standards were used to interpolate median fluorescence intensity values of each cytokine in each sample into a pg/ml value per the manufacturer’s instructions before adjusting for dilution factors and normalizing to background.

To prepare splenocyte samples, harvested spleens were collected in 10 ml HBSS on ice before manual digestion through a 70-μm nylon mesh strainer. Cells were centrifuged and resuspended in lysis buffer (150 mM ammonium chloride [NH₄Cl], 0.7 mM potassium phosphate monobasic [KH₂PO₄] in dH₂O) for 4 min at room temperature with agitation. HBSS was added to samples before centrifugation, counting, and resuspension at 5 × 10⁶ cells/ml in ice-cold FACS buffer (1% FBS, 1 mM EDTA in PBS). For all experiments, 500 μl cell suspension was added to 12 × 75–mm polystyrene tubes and incubated with 100 μl TruStain FcX (BioLegend, San Diego, CA) on ice for 20 min. The following Abs were used for labeling cells: PE rat anti-mouse CD45 (clone 30-F11; BioLegend), FITC rat anti-mouse CD3 (clone 17A2; BD Biosciences, San Jose, CA), and allophycocyanin rat anti-mouse CD4 (clone RM4-5; BD Biosciences). A quantity of 50 μl of each Ab was added to samples and incubated on ice in the dark for 1 h. Cells were fixed using BD Cytofix Fixation Buffer (BD Biosciences) according to the manufacturer’s instructions and stored at 4°C protected from light before acquisition on a FACSCalibur cytometer equipped with CellQuest Pro software (BD Biosciences) within 1 wk of fixation. Analysis was performed using FlowJo version 10.8.0 (BD Biosciences).

Statistical analyses of weights and titers were carried out using GraphPad Prism version 9.2.0. Unpaired, two-tailed Welch’s t tests were performed to determine the significance of differences in endpoint titers between groups, as well as in cytokine contents in BALF and serum. In all cases, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p < 0.0001.

We recently reported that i.n. administration of RICs to mice induces a rapid and durable serum anti-RT IgG response within 10–14 d without a detectable IgM phase. To ascertain whether CD4⁺ T cells are involved in this response, groups of adult BALB/c mice were treated with an anti-CD4 mAb (400 µg by i.p. injection) before (d −1) and after (d +1, +4) i.n. RIC administration on study day 0. The anti-CD4 Ab regimen resulted in a near-complete of depletion of CD4⁺ T cells, as determined in a parallel group of mice (Fig. 1A, 1B). RT-specific serum IgG titers were measured in sera collected on days 7, 14, and 30 following RIC administration. In the control group of mice, i.n. RIC administration resulted in the onset of RT-specific serum IgG by day 14, with reciprocal endpoint titers exceeding 10³ by day 30 (Fig. 1C). Although IgG subclass profiles were not determined in this study, we previously demonstrated that RIC vaccination elicits an IgG1-dominated response (17). By comparison, in mice treated with the anti-CD4 mAb, RT-specific IgG levels were at baseline, even on day 30 (Fig. 1C). This result demonstrates the necessity of CD4⁺ T cells in the humoral response to RICs and specifically warrants an investigation into the role of T follicular helper cells in this process, especially in the draining lymph nodes (22).

We next sought to investigate the impact of RIC exposure on local inflammatory cytokine and chemokine levels, reasoning that the nature of the response may provide a clue to the underlying factors that drive durable anti-toxin B cell responses. To address this question, we collected serum and BALF from mice 6, 12, and 18 h after i.n. RIC (20 µg PB10, 20 µg SylH3, 1 µg RT) administration and then subjected samples to a 32-plex mouse cytokine/chemokine array. We also collected samples from groups of mice treated i.n. with just RT (1 µg), the PB10/SylH3 mAb mixture (20 µg each mAb) alone, or vehicle (PBS). The data presented in this study represent the combined average concentrations and fold changes of two independent replicates. In each replicate, there were three mice per group. Therefore, the averages presented in Fig. 2 and Supplemental Table I constitute six animals per treatment at any given time point.

Although PBS did not induce any notable changes in inflammatory cytokines in either serum or BALF, the PB10/SylH3 mAb mixture did transiently stimulate a variety of cytokines within the panel, although the changes were not statistically significant (Supplemental Table I). BALF samples were more strongly impacted than serum samples, suggesting changes may be associated with local FcR activation.

On the other hand, i.n. delivery of RT stimulated the secretion of a variety of proinflammatory cytokines and chemokines in both serum and BALF to levels that were orders of magnitude higher than background, including G-CSF, IL-6, KC (CXCL1), MCP-1, and MIP-1β (Supplemental Table I). Those same analytes (except KC) were detected in serum of nonhuman primates exposed to RT (8). In the mice, upticks in cytokine and chemokine levels following RT exposure were generally evident in the BALF first and then in serum, consistent with the diffusion of toxin and toxin-induced inflammation from the lung mucosa (Fig. 2). IL-6 stood out because its levels increased >1500-fold in BALF as compared with control animals. IL-6 has previously been implicated as a driver of RT-induced lung pathophysiology in both murine and nonhuman primate models of aerosolized RT exposure (8, 11, 23). Intranasal delivery of RICs stimulated a largely muted inflammatory response in the BALF and serum, as compared with RT itself, apart from KC and IL-6 (Fig. 2; Supplemental Table I). For example, IL-6 levels increased 135-fold in BALF at 12 h following RIC exposure without a corresponding increase in serum IL-6 levels. Collectively, these results suggest that inflammatory responses following RICs are not only are dampened relative to RT but are confined to the lung environment.

Elevated levels IL-6 in the BALF following RIC exposure could contribute to the observed onset of RT-specific IgG by multiple mechanisms, including B cell and T follicular helper cell differentiation (24). To address the role of IL-6 in our model, BALB/c mice were treated with an IL-6–neutralizing mAb (clone MP5-20F3, Bio X Cell) prior to and following RIC immunization. The IL-6–neutralizing mAb regimen resulted in undetectable levels of IL-6 in serum and a significant, albeit partial, reduction in IL-6 levels in BALF (Supplemental Fig. 1A, 1B). However, the IL-6–neutralizing mAb treatment had no impact on the kinetics or magnitude of RT-specific serum IgG levels following RIC treatment as compared with control mice (Supplemental Fig. 1C). Although these results suggest that IL-6 may not be necessary for the onset of RT-specific Abs following RIC exposure, we cannot exclude the possibility that the reduced amounts of IL-6 detected in the BALF are sufficient to promote toxin-specific Ab responses. Further studies aimed at complete ablation of IL-6 in the context of RIC exposure will be needed to resolve the role of this cytokine in inducing downstream RT-specific Ab responses.

Abs in mucosal secretions of the upper and lower airways play a central role in immunity and tissue homeostasis by preventing the uptake of allergens, toxins, and pathogenic agents (25). Indeed, there are multiple mechanisms within the airways to purge Ag-Ab complexes, including mucociliary clearance and phagocytosis by alveolar macrophages. However, there is evidence to suggest that Ab-Ag complexes may also be sampled by APCs within the lung and/or draining lymph nodes with consequences for both mucosal and systemic immunity. Specifically, we reported that, in mice, i.n. delivery of RICs elicits the rapid onset of high-titer, toxin-neutralizing IgG Abs that persist for months (17). We have now extended those original observations and demonstrated that such responses are T cell dependent and occur in the context of elevated levels of IL-6 and possibly other proinflammatory cytokines and chemokines. Although many questions remain regarding the pathways that result in the onset of toxin-specific B cells, the elevated levels of proinflammatory cytokines/chemokine in BALFs following RIC exposure are consistent with the local sampling of ICs.

Prior to this work, the role of T cell help in mediating the humoral response to RICs was unclear. Although the appearance of anti-RT IgG within days following RIC exposure was evidence of B cell class switching and hinted at T cell involvement, the kinetics of the response were more characteristic of a T-independent reaction (17). We now conclude that CD4⁺ T cells are not only involved but essential for mounting a humoral response to RIC vaccination. The necessity of T cell help is particularly interesting, because previous work reported endogenous IgG generation within roughly 7 d of i.n. RIC administration. This rapid timeline for a T-dependent response is unusual, because T cell activation alone can take 24 h, and germinal centers typically do not begin forming until ∼4 d after initial Ag exposure (26) with full formation ∼7–10 d after Ag encounter (27). Thus, i.n. administration of RICs stimulates an extremely fast T-dependent humoral response.

This observation raises the question of where RICs are being presented in the context of the upper and/or lower airways. Canonically, local APCs migrate to the draining lymph node to present Ag to T cells. However, a growing body of literature suggests that Ag presentation may occur within the lung mucosa (28). For example, a recent study implicated interstitial macrophages in the uptake and local presentation of Ag in a murine model of OVA-induced allergic asthma (29). A role for macrophages in RIC sampling would not be surprising, considering that RT itself is efficiently internalized via the mannose receptor and possibly other C-type lectins (4, 30). Macrophages including lung-derived macrophages also secrete an array of proinflammatory cytokines, including IL-6, in response to RT (6, 7). The same could hold true for RICs, because we have shown that PB10 and SylH3 do not necessarily preclude RT’s ability to adhere to lung macrophages (11). Whether macrophages discriminate between RT and RICs is an interesting question, especially considering recent studies demonstrating that virus-Ab complexes elicit different degrees of inflammasome activation and inflammatory chemokines/cytokines compared with virus alone (31).

The potential of ICs as platforms for systemic and mucosal vaccination has largely been viewed through the lens of augmenting Ag uptake and presentation by promoting Fc receptor interactions (16). In this respect, RICs are different in that PB10 and SylH3 likely function as a means of attenuating RT rather than enhancing FcγR-mediated uptake. For example, we reported that RICs generated with PB10 and SylH3 LALAPG derivatives, which are unable to engage with FcγRs, were as immunostimulatory as PB10 and SylH3 IgG1 (17). Similarly, RIC immunostimulatory activity was unaffected in FcγR-deficient mice. The fact that the proinflammatory cytokine/chemokine profile of RICs resembles a damped-down version of RT is also consistent with PB10 and SylH3 serving not to redirect RT specific APCs but to attenuate RT such that it retains its intrinsic antigenicity without cytotoxicity. As appealing a model as that is, it is not the whole story, because the kinetics of de novo RT Ab onset following RIC exposure are distinct from those elicited by low-dose RT (17). Sorting the intrinsic and extrinsic factors that underlie the mucosal immunogenicity of RICs has potential application to mucosal adjuvants and vaccines.

The authors have no financial conflicts of interest.

We thank members of the Wadsworth Center’s Veterinary Sciences team for caring for our animals. We also thank Dr. Renjie Song of the Wadsworth Center’s Immunology Core for assistance in flow cytometry experiments and Grace Freeman-Gallant for her guidance in the completion of the Luminex assays.