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

Abstract

The underlying contribution of immune complexes in modulating adaptive immunity in mucosal tissues remains poorly understood. In this report, we examined, in mice, the proinflammatory response elicited by intranasal delivery of the biothreat agent ricin toxin (RT) in association with two toxin-neutralizing mAbs, SylH3 and PB10. We previously demonstrated that ricin-immune complexes (RICs) induce the rapid onset of high-titer toxin-neutralizing Abs that persist for months. We now demonstrate that such responses are dependent on CD4+ T cell help, because treatment of mice with an anti-CD4 mAb abrogated the onset of RT-specific Abs following intranasal RICs exposure. To define the inflammatory environment associated with RIC exposure, we collected bronchoalveolar lavage fluid (BALF) and sera from mice 6, 12, and 18 h after they had received RT or RICs by the intranasal route. A 32-plex cytometric bead array revealed an inflammatory profile elicited by RT that was dominated by IL-6 (>1500-fold increase in BALF) and secondarily by KC (CXCL1), G-CSF, GM-CSF, and MCP-1. RICs induced inflammatory profiles in both BALF and serum response that were similar to RT, albeit at markedly reduced levels. These results demonstrate that RICs retain the capacity to induce local and systemic inflammatory cytokines/chemokines that, in turn, may influence Ag sampling and presentation in the lung mucosa and draining lymph nodes. A better understanding of the fate of immune complexes following intranasal delivery has implications for the development of mucosal vaccines for biothreats and emerging infectious diseases.

FIGURE 1.

Role of CD4⁺ T cells in mounting a humoral response following i.n. RIC immunization. RICs were administered i.n. alone or in tandem with anti-CD4 treatment. (A) Combined population of Th cells (CD3⁺CD4⁺) in the spleens of untreated mice (n = 3). (B) Combined population of Th cells in the spleens of mice treated with anti-CD4 Ab 24 h prior to harvest (n = 3). (C) Anti-RT serum IgG titers of RIC-immunized mice receiving no additional treatment or anti-CD4 Ab (n = 6) or control animals receiving only anti-CD4 Ab or PBS (n = 4). Anti-CD4 treatment mice received anti-CD4 mAb 24 h prior to RIC vaccination, as well as 1 and 4 d postimmunization to ensure continual depletion. Day −7 is shown as a negative control time point prior to any RIC or anti-CD4 treatments. Significance of endpoint titers was determined by unpaired, two-tailed Welch’s t test. ****p < 0.0001.

FIGURE 2.

RICs induce an intermediate inflammatory environment within the respiratory tract. RIC, RT, or PBS was administered to mice by the i.n. route before harvest of serum and BALF at 6, 12, or 18 h post-treatment for cytokine content analysis. For each treatment at each time point, n = 6. Concentrations are expressed as fold change over PBS-treated samples. Notable inflammatory cytokines produced in high quantities in both (A) serum and (B) BALF. Cytokines with a nonzero fold change at any tested time point are shown in (C) for serum samples and (D) for BALF samples. Scales for fold change heatmaps in (A)–(D) vary. Mean group concentration values for all samples, including those with no change, and their statistical significance relative to control samples are presented in Supplemental Table I. Special attention should be given to the varying scales associated with the heatmaps. Significance of concentrations and fold change for each cytokine and sample type among RT- and RIC-treated mice were determined by unpaired, two-tailed Welch’s t tests, using cytokine concentrations of PBS-treated animals as baseline values.