Genetic control of susceptibility to candida albicans in SM/J mice

In the immunocompromised host, invasive infection with the fungal pathogen Candida albicans is associated with high morbidity and mortality. Sporadic cases in otherwise normal individuals are rare, and they are thought to be associated with genetic predisposition. Using a mouse model of systemic infection with C. albicans, we identified the SM/J mouse strain as unusually susceptible to infection. Genetic linkage studies in informative [C57BL/6JxSM/J]F2 mice identified a major locus on distal chromosome 15, given the appellation Carg5, that regulates C. albicans replication in SM/J mice. Cellular and molecular immunophenotyping experiments, as well as functional studies in purified cell populations from SM/J and C57BL/6J, and in [C57BL/ 6JxSM/J]F2 mice fixed for homozygous or heterozygous Carg5 alleles, indicate that Carg5-regulated susceptibility in SM/J is associated with a complex defect in the myeloid compartment of these mice. SM/J neutrophils express lower levels of Ly6G, and importantly, they show significantly reduced production of reactive oxygen species in response to stimulation with fMLF and PMA. Likewise, CD11b+Ly6G2Ly6Chi inflammatory monocytes were present at lower levels in the blood of infected SM/J, recruited less efficiently at the site of infection, and displayed blunted oxidative burst. Studies in F2 mice establish strong correlations between Carg5 alleles, Ly6G expression, production of serum CCL2 (MCP-1), and susceptibility to C. albicans. Genomic DNA sequencing of chromatin immunoprecipitated for myeloid proinflammatory transcription factors IRF1, IRF8, STAT1 and NF-kB, as well as RNA sequencing, were used to develop a "myeloid inflammatory score" and systematically analyze and prioritize potential candidate genes in the Carg5 interval.Peer reviewed: YesNRC publication: Ye

Relative comparative <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA load in spleen and liver of immunized mice.

<p>To compare relative load of <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA in liver and spleen, BALB/c (3 mice per group) were immunized iv on tail with 1×10⁵ iSPZ (<i>wt</i> or <i>spect (</i>−<i>)</i>) in 500 µl of RPMI. Two hours later, a real-time PCR was performed following extraction of respective parasite RNA. Every sample was done in duplicate. To avoid any contamination by eventual parasite from blood, the liver and spleen were perfused with PBS before performing the RT-PCR. Figures <b>a</b> and <b>b</b> represent <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA load, respectively, in the liver and spleen 2 h after iSPZ injection (iv). Naïve mice (receiving only 500 µl iv of DMEM) were used as a control.</p

Infected hepatocytes present a <i>Pb</i>CSP-specific epitope to primed CD8+ T-cells and protect mice against SPZ challenge.

<p>Each recipient <i>TAP−/−</i> mouse (H-2K^b) received 7×10⁵<i>Pb</i>SPZ-infected BALB/c (H-2k^d) hepatocytes by IS transfer as described in Materials and Methods. Before IS injection, infected hepatocytes were isolated from BALB/c mice that were injected (iv) with 10⁶ANKA <i>wt Pb</i>SPZ 2 h earlier. C7 and S14 (20 million cells per mouse, iv) were injected into the corresponding group 4 h after IS transfer. Mice were protected if they remained parasite negative 2 weeks after infected hepatocyte transfer.</p

Frequency of <i>Pb</i>CSP-specific CD8+ T-cells in different organs of BALB/c mice immunized with <i>wt</i> or <i>spect (</i>−<i>)</i> iSPZ.

<p>BALB/c mice (4 mice per group) were immunized (iv) with <i>wt</i> or <i>spect (</i>−<i>)</i> ANKA <i>Pb</i>iSPZ (one dose of 1×10⁵ iSPZ). Seven (7) days later, PBL, liver and spleen cells of mice were isolated to determine frequency of <i>Pb</i>CSP-specific CD8+ T-cells by flow cytometry (FACScan). The CD8+ T-cells were double stained with PE-conjugated <i>Pb</i>CSP epitope tetramer and FITC-conjugated anti-mouse CD8b antibody. The naive group received neither <i>wt</i> nor <i>spect (</i>−<i>)</i> iSPZ. p-value compares statistically significant mean of CD8+ T-cell frequency between <i>wt</i> and <i>spect (</i>−<i>)</i> iSPZ-immunized groups.</p

Specificity of activation of C7 clone and protection<b>.</b>

<p>Mice were first injected (iv) or not with 10³ live wild-type (<i>wt</i>) ANKA <i>Pb</i>SPZ 10 h before they received or not 7×10⁵ naïve-BALB/c hepatocytes and C7 clone as indicated above. Infected hepatocytes were isolated from BALB/c mice injected with 10⁶ ANKA <i>wt Pb</i>SPZ 2 h earlier; and naïve BALB/c hepatocytes were isolated from naïve BALB/c mice. Mice were considered protected if they remained parasite negative 2 weeks after infection.</p

BALB/c mice injected with iSPZ-loaded hepatocytes are protected against SPZ challenge.

<p>BALB/c mice were injected (IS transfer) with 7×10⁵<i>Pb</i>iSPZ-infected or naïve BALB/c hepatocytes. Infected hepatocytes were obtained from BALB/c mice immunized with 10⁶ ANKA <i>wt</i> iSPZ 2 h earlier. Recipient mice were then challenged with two different doses (2×10³ and 5×10³, respectively, A and B) of live <i>Pb</i>SPZ one week later. Mice were considered protected if they remained parasite negative 2 weeks after challenge.</p