Border patrol gone awry: Lung NKT cell activation by Francisella tularensis exacerbates tularemia-like disease

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia--because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of NKT cells within the lung interstitium. Activated NKT cells produced interferon-γ and promoted both local and systemic proinflammatory responses. Consistent with these results, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice

CD1d^-/- mice exhibit a tempered inflammatory response to LVS.

<p>Cytokine levels were measured at 7d p.i. in lung homogenates <b>(A)</b> or serum <b>(B)</b> by CBA. Data are combined from at least 3 experiments with 10–20 mice/group. Bars are mean+SD. Comparisons in these experiments were made as indicated in Materials and Methods. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

CD1d^-/- mice have less severe neutrophilia after intranasal LVS infection.

<p><b>(A)</b> AST and ALT levels were measured in serum of infected mice on d7 p.i. Data are combined from 3 experiments (<i>n</i> = 10–12). <u>Mean+SD</u>. Shaded regions denote reference range. <b>(B)</b> CBC analysis of blood from LVS-infected mice performed on d7. Data are combined from 2 separate experiments (<i>n</i> = 15 mice/group). Bars are mean+SD. <b>(C)</b> Percent neutrophils in blood at the indicated time points p.i. Data are combined from 2 separate experiments. Bars are mean+SD of 5–15 mice/group/time point. <b>(D)</b> On d7 serum and lung G-CSF levels were measured by CBA. Data are combined from 2 experiments (<i>n</i> = 15 mice/group). Bars are mean+SD. Comparisons were made as indicated in Materials and Methods. *<i>p</i><0.05, ***<i>p</i><0.001.</p

CD1d^-/- mice are less susceptible to i.n. LVS infection.

<p><b>(A)</b> Survival of B6 or Jα18^-/- mice after i.n. LVS infection. Survival of Jα18^-/- and WT mice after intranasal LVS infection (8000 cfu). Curves were compared using Log-rank (Mantel-Cox) Test. Data are from one of three similar experiments with at least 10 mice/group. <b>(B)</b> B6 or CD1d^-/- mice were infected i.n. with LVS and monitored daily for weight loss. The mean (±SD) weight of 50 animals/group in several independent experiments following intranasal infection (Accumulation of five independent experiments with 10 mice/group in each experiment) is presented. <b>(C)</b> Clinical score at various time points p.i. as determined in Materials and Methods. Data are cumulative from four similar experiments (mean+SD) with 5–10 mice/group/time point. Data were analyzed as indicated in Materials and Methods. <b>(D)</b> Survival of B6 or CD1d^-/- mice after i.n. LVS infection. Cumulative survival curves from 4 experiments with 40 mice/group. Curves were compared using Log-rank (Mantel-Cox) Test. **<i>p</i><0.01, ***<i>p</i><0.001.</p

CD1d^-/- mice have modestly reduced lung LVS burden but no differences in lung pathology.

<p><b>(A)</b> Lung burden was determined at various times p.i. as described in Materials and Methods. Data are combined from at least 3 separate experiments. Bars are mean+SD of 10–15 mice/group/time point. <b>(B)</b> Oxygen saturation was measured as described in Materials and Methods. Data are cumulative of 3 separate experiments with 5–10 mice/group (mean±SD). <b>(C)</b> H&E stained formalin fixed paraffin embedded (FFPE) lung sections d9 p.i. 40X (top) or 400X (bottom) magnification with scale bars as indicated. Data are representative of three mice per group. Arrows indicate aggregations of lymphocytes. Data were analyzed as indicated in Materials and Methods. **<i>p</i><0.01, ***<i>p</i><0.001.</p

Formation of iBALT in CD1d^-/- mice after i.n. LVS infection.

<p><b>(A)</b> Groups of B6 or CD1d^-/- mice were infected i.n. with LVS. On d7 p.i., lungs were analyzed by flow cytometry for the indicated cell populations. Data are cumulative of 2 experiments with 9–10 mice/group. Bars are mean+SD; compared as indicated in Materials and Methods. <b>(B)</b> Representative images from <i>F</i>. <i>tularensis</i> LVS-infected lungs d7 p.i. Images are individual mice from 2 of 3 similar experiments. 20X magnification; bars are 80 μm. Dashed lines indicate large airways as identified by autofluorescence of epithelial cells. Arrows indicate rudimentary iBALT formation in B6 lung and well-developed iBALT in lungs of CD1d^-/-mice. <b>(C)</b> Enumeration of iBALT from d7 infected lung sections. Data are cumulative from three experiments (<i>n</i> = 6 mice/group). Bars are mean+SD. Data were compared using Mann Whitney U test. <b>(D)</b> DC subsets in infected lungs were identified as described in Experimental Procedures. Representative results from 1 of 3 similar experiments (<i>n</i> = 5 mice/group). Bars are mean+SD; Mann Whitney U test. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

CD1d^-/- mice have reduced splenic LVS burden but no difference in the liver.

<p><b>(A)</b> Blood, liver, and spleen burden were determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004975#ppat.1004975.g004" target="_blank">Fig 4</a>. Data are combined from 3 separate experiments. Bars are mean+SD of 6–10 mice/group/time point. Data were analyzed as indicated in Materials and Methods. **<i>p</i><0.01. <b>(B)</b> H&E stained FFPE liver (top) and spleen (bottom) sections d9 p.i., 200X magnification. Data are representative of 3 mice/group. Arrows indicate granulomas.</p

NKT cell activation by LVS is TCR-dependent.

<p><b>(A)</b> Representative plots of NKT cell localization, activation, and IFN-γ production after i.n. LVS infection of Nur77^gfp mice. Numbers in contour plots are % NKT cells. Numbers in histograms are median fluorescence of infected mice (open histograms) and mock-infected controls (shaded histograms). Data are representative of three similar experiments (<i>n</i> = 10). <b>(B)</b> Percent IFN-γ+ NKT cells in each compartment after LVS infection. Bars are mean+SD. Data are representative from one of three similar experiments (<i>n</i> = 3 mice). Comparison was made by one-way ANOVA with Tukey’s posttest. ***<i>p</i><0.001.</p