Conditional Stat1 Ablation Reveals the Importance of Interferon Signaling for Immunity to Listeria monocytogenes Infection

Signal transducer and activator of transcription 1 (Stat1) is a key player in responses to interferons (IFN). Mutations of Stat1 cause severe immune deficiencies in humans and mice. Here we investigate the importance of Stat1 signaling for the innate and secondary immune response to the intracellular bacterial pathogen Listeria monocytogenes (Lm). Cell type-restricted ablation of the Stat1 gene in naïve animals revealed unique roles in three cell types: macrophage Stat1 signaling protected against lethal Lm infection, whereas Stat1 ablation in dendritic cells (DC) did not affect survival. T lymphocyte Stat1 reduced survival. Type I IFN (IFN-I) signaling in T lymphocytes reportedly weakens innate resistance to Lm. Surprisingly, the effect of Stat1 signaling was much more pronounced, indicating a contribution of Stat1 to pathways other than the IFN-I pathway. In stark contrast, Stat1 activity in both DC and T cells contributed positively to secondary immune responses against Lm in immunized animals, while macrophage Stat1 was dispensable. Our findings provide the first genetic evidence that Stat1 signaling in different cell types produces antagonistic effects on innate protection against Lm that are obscured in mice with complete Stat1 deficiency. They further demonstrate a drastic change in the cell type-dependent Stat1 requirement for memory responses to Lm infection

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

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

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

Delayed cytokine response in Ifnar1−/− compared to Wt mice after intragastric infection with <i>Listeria monocytogenes</i>.

<p>A.Cytokine mRNA expression in the spleens of C57BL/6N (solid line) or Ifnar1−/− mice (hatched line) 24 h, 48 h and 72 h after infection with 5×10∧9 CFU of strain LO28InlA* by intragastric gavage (i.g.), or 1×10∧6 LO28InlA* by intraperitoneal infection (i.p.) was determined by qPCR. mRNAs were normalized to the GAPDH housekeeping control and values obtained from infected Wt mice set to 1. Data from Ifnar1−/− mice thus represent the relative induction compared to the value obtained at the same time point from Wt animals. Mean values and SEM from 9 mice (spleen, all time points of i.g. infection), 7 mice (spleen after 24 h of i.p. infection) or 4 mice (spleen, 48 h and 72 h after i.p. infection) are indicated. B. Serum IFNγ levels of C57BL/6N Wt and Ifnar1−/− mice infected i.g. for 24 h, 48 h or 72 h with 5×10∧9 CFU of strain LO28InlA* by intragastric gavage (i.g.), or with 1×10∧6 CFU LO28InlA* by intraperitoneal injection were determined using a flow cytometry-based bead array. Mean values and SEM from 9 mice (24 h and 48 h time points) or 12 mice (72 h time point) are indicated.</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

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