Route of Infection Determines the Impact of Type I Interferons on Innate Immunity to <i>Listeria monocytogenes</i>

<div><p><i>Listeria monocytogenes</i> is a food-borne pathogen which causes mild to life threatening disease in humans. Ingestion of contaminated food delivers the pathogen to the gastrointestinal tract, where it crosses the epithelial barrier and spreads to internal organs. Type I interferons (IFN-I) are produced during infection and decrease host resistance after systemic delivery of <i>L. monocytogenes</i>. Here we show that mice benefit from IFN-I production following infection with <i>L. monocytogenes</i> via the gastrointestinal route. Intragastric infection lead to increased lethality of IFN-I receptor chain 1-deficient (Ifnar1−/−) animals and to higher bacterial numbers in liver and spleen. Compared to infection from the peritoneum, bacteria infecting via the intestinal tract localized more often to periportal and pericentral regions of the liver and less frequently to the margins of liver lobes. Vigorous replication of intestine-borne <i>L. monocytogenes</i> in the livers of Ifnar1−/− mice 48 h post infection was accompanied by the formation of large inflammatory infiltrates in this organ and massive death of surrounding hepatocytes. This was not observed in Ifnar1−/− mice after intraperitoneal infection. The inflammatory response to infection is shaped by alterations in splenic cytokine production, particularly IFNγ, which differs after intragastric versus intraperitoneal infection. Taken together, our data suggest that the adverse or beneficial role of a cytokine may vary with the route of infection and that IFN-I are not harmful when infection with <i>L. monocytogenes</i> occurs via the natural route.</p></div

Primer sequences used for qPCR cytokine-induced genes.

<p>Primer sequences used for qPCR cytokine-induced genes.</p

Localization and replication of <i>Listeria monocytogenes</i> in the intestinal tract and stimulation of cytokine production in response to infection.

<p>A, upper panels. Anti-Listeria serum was used to detect Lm in the intestinal mucosa from C57BL/6N Wt or Ifnar1−/− mice 48 h post infection. Listeria infection, in both Wt and Ifnar1−/− mice occurred mostly in mucosal tissue beneath the epithelial layer. A, lower panels. Anti-Listeria serum was used to detect Lm in Wt or Ifnar1−/− Peyer's patches (PP) 48 h post infection. B, C. Bacterial numbers in PP (B) at day 2 or mesenteric lymph nodes (MLNs, C) over three days, determined by CFU assay. Standard variations for MLN indicate the median with interquartile range from 7 mice (24 h and 48 h time points) or 12 mice (72 h time point). D. Analysis of Peyer's patch mRNAs by qPCR at the indicated times after infection. Mean values and SEM from 9 mice per time point are indicated. All experiments were performed with C57BL/6N Wt and Ifnar1−/− mice infected with a dose of 5×10∧9 CFU of the LO28InlA* strain by intragastric gavage (i.g.).</p

Comparison of the CD45+ fraction of nonparenchymal liver cells (NPC) of i.g.- and i.p.-infected C57BL/6N Wt and Ifnar1−/− mice.

<p>A–D. CD45+ nonparenchymal liver cells (NPC) from infected mice were analyzed for neutrophils (A), macrophages (B), inflammatory monocytes (C) and T cells (D) using the indicated markers 48 h post infection. The data are representative of three different experiments with four mice in each group. I.p. infections were performed with doses of 1×10∧6 CFU and i.g. infections with 5×10∧9 CFU of LO28InlA*.</p

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer-1

-oocyte mtDNA) and ARMS-qPCR (non-discriminative (ND) and discriminative (D) assays) for the low-abundant SNP variant (target here: donor mtDNA) at the mutated site. The amplification efficiency of the D assay was 91%. Without further optimization concerning conditions for enzymatic digestion, quantification by REMS-qPCR targeting a single donor-B-specific SNP reached a detection limit of 0.02%, i.e. a point mutation discrimination selectivity factor of 5 × 10. It allowed detection of heteroplasmy (0.1%) in the donor B-derived clone CB2. This could not be detected by conventional ARMS-qPCR discriminating point mutations only down to 0.1% (see: illustrated improvement of discrimination). For clarity each plot is presented as the mean calculated from duplicate amplification reactions. Individual Ct values, i.e. PCR cycle numbers at which plots crossed an arbitrarily placed signal threshold, are given in the figure key. An independent technical replicate of this REMS-qPCR experiment demonstrated reproducibility of the method (data not shown).<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer"</p><p>http://www.biomedcentral.com/1471-213X/7/141</p><p>BMC Developmental Biology 2007;7():141-141.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2323970.</p><p></p

Low infectious doses or dissemination via infected cells do not alter the adverse effect of IFN-I after systemic infection with <i>Listeria monocytogenes</i>.

<p>A, B,.Doses of10∧2 and 10∧4 Lm were injected intravenously (i.v.) into C57BL/6N Wt and Ifnar1−/− mice and bacterial loads in spleens (A) and livers (B) were determined by CFU assay 72 h after infection. C, D. Wt bone marrow-derived macrophages or myeloid dendritic cells were infected <i>in vitro</i> with a MOI of 10 for 1 h, vigorously washed in PBS and 10∧4 cells were injected i.v. into C57BL/6N Wt and Ifnar1−/− mice. The injected populations contained 3–5×10∧3 viable Lm. Bacterial loads in spleens (C) and livers (D) were measured by CFU assay 72 h after infection.</p

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer-3

-oocyte mtDNA) and ARMS-qPCR (non-discriminative (ND) and discriminative (D) assays) for the low-abundant SNP variant (target here: donor mtDNA) at the mutated site. The amplification efficiency of the D assay was 91%. Without further optimization concerning conditions for enzymatic digestion, quantification by REMS-qPCR targeting a single donor-B-specific SNP reached a detection limit of 0.02%, i.e. a point mutation discrimination selectivity factor of 5 × 10. It allowed detection of heteroplasmy (0.1%) in the donor B-derived clone CB2. This could not be detected by conventional ARMS-qPCR discriminating point mutations only down to 0.1% (see: illustrated improvement of discrimination). For clarity each plot is presented as the mean calculated from duplicate amplification reactions. Individual Ct values, i.e. PCR cycle numbers at which plots crossed an arbitrarily placed signal threshold, are given in the figure key. An independent technical replicate of this REMS-qPCR experiment demonstrated reproducibility of the method (data not shown).<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer"</p><p>http://www.biomedcentral.com/1471-213X/7/141</p><p>BMC Developmental Biology 2007;7():141-141.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2323970.</p><p></p

Localization of inflammatory infiltrates in livers of i.p. vs i.g. infected mice.

<p>A. Localization of bacteria-containing infiltrates to the margins of the liver lobes (black) or to the periportal or pericentral region (white). The graph indicates relative numbers determined in five C57BL/6N Wt and Ifnar1−/− mice 48 h post i.p. or i.g. infection. The anti-Listeria staining in the panel on the right indicates marginal and periportal infiltrates from a representative Wt sample 48 h post infection. B. Histochemical analysis of hematoxylin-stained liver sections obtained 24 h, 48 h or 72 h after i.g. and 48 h or 72 h after i.p administration of strain LO28InlA* to C57BL/6N or Ifnar1−/− mice. C, D. Gr1+ (left panels) or TUNEL+ cells (right panels) in inflammatory liver infiltrates. Liver sections obtained 48 h (C) or 72 h (D) after i.g administration of strain LO28InlA* to Ifnar1−/− mice were stained with antibody to Gr1 or subjected to TUNEL staining. GR1+ and TUNEL+ cells appear red. E. Serum ALT levels from i.g.-infected mice. The data are representative of two different experiments with seven mice in each group and time point.</p

Infection and infection-induced death of splenocytes from i.p.- or i.g.- infected C57BL/6N Wt and Ifnar1−/− mice.

<p>Representative samples obtained 48 h after i.p. administration of 1×10∧6 CFU of strain LO28InlA* (upper panels) or i.g. administration of 5×10∧9 LO28InlA* (lower panels) to C57BL/6N Wt or Ifnar1−/− mice as indicated. Panels on the left were stained with anti-Lm antibody. Panels on the right were subjected to in situ TUNEL assay. Infected cells and TUNEL+ cells appear red. The orange background seen in the red pulp of some sections is caused by erythrocyte haemoglobin. The bar graphs show quantification of TUNEL+ cells from sections of four spleens of i.p-infected and six spleens of i.g-infected C57BL/6N (black bar) and Ifnar1−/− (grey bar) mice.</p

IFN-I increase host resistance after intragastric infection with <i>Listeria monocytogenes</i>.

<p>C57BL/6N Wt and Ifnar1−/− mice were infected with Lm strain LO28InlA*. A, B. Numbers of bacteria in livers (A) and spleens (B) were determined by CFU assay 72 h after intraperitoneal (i.p.) infection with 1×10∧6 Lm. C, D. Bacterial loads of livers (C) and spleens (D) were examined by CFU assay 72 h after intragastric gavage (i.g.) with 5×10∧9 Lm. Plots indicate the Median of bacterial counts. E. 14 mice per group were infected i.g. with 5×10∧9 Lm LO28InlA* and survival was monitored over ten days.</p