Changes in mammary bacterial counts post-IMI with <i>E. coli</i> or <i>S. aureus</i>.

<p>At 12 h and 24 h post-IMI, mammary tissue was harvested and evaluated for bacterial growth. (P<0.01 **, P<0.001***)</p

<i>In vivo</i> mammary NF-kappaB activity post-IMI with <i>E. coli</i> compared to <i>S. aureus</i>.

<p>(A) <i>In vivo</i> imaging of local NF-kappaB activity in mammary glands (ventral view) showed a transient and fast activation for both pathogens compared to sham-inoculated (PBS) glands and also about a 3-fold higher activation post-MI with <i>E. coli</i> compared to <i>S. aureus</i>. Data represent the flux density (total flux radiance per selected body area (in p*(s*m²))) as a quantitative measure of the bioluminescent signal correlating with the NF-kappaB activity. (B) A representative photograph of lactating mice (at 0 h, most left) compared to mice at 6 h post-IMI with sham (PBS, 2nd from left), at 6 h post-IMI with <i>S. aureus</i> (3rd from left), or at 6 h post-IMI with <i>E. coli</i> (most right). The intensity of luminescence was scaled based on the radiance (in p*(s*m²)). (C) Mammary glands of NF-kappaB reporter mice were excised at 12 h and 24 h post-IMI and their <i>ex vivo</i> luminescence was measured. The graph represents the ratio between the total flux radiance per area (in p*(s*m²)) at 4 h of the inoculated gland and the value from the non-injected 3th gland in the same mouse. Although the maximal activation occurred earlier, the local NF-kappaB activity was still significantly higher in <i>E. coli</i> compared to <i>S. aureus</i> mammary glands at both these time points of relevance for cytokine transcription. (D) Representative photographical view of excised mammary glands post-IMI with <i>E. coli</i> (top), <i>S. aureus</i> (middle) or after sham-inoculation (PBS, bottom). Letters of homogeneous subsets were marked if the difference between treatments were statistically significant (P<0.05).</p

Differential nuclear translocation of mammary NF-kappaB p65, immune cell influx and damage at the mammary epithelium post-IMI with <i>S. aureus</i> compared to <i>E. coli</i>.

<p>(A) At 12 h post-IMI, a nuclear translocation of NF-kappaB subunit p65 was observed in the epithelia of both <i>E. coli</i> (-1a-) and <i>S. aureus-</i>infected glands (-4a-). At this early time point, a strong cytoplasmatic NF-kappaB p65 signal was only seen post-IMI with <i>E. coli</i>. At 24 h post-IMI, NF-kappaB p65 was mainly detected in the cytoplasm in the mammary epithelium of both <i>E. coli-</i>(-6-) and <i>S. aureus-</i>infected glands (-10-). while at this time both sham-inoculated (PBS, -11-) and lactating (at 0 h,-12-) glands displayed very low basal levels of latent expressed cytoplasmic NF-kappaB p65. At 12 h post-IMI with <i>E. coli</i> the polymorphonuclear cells in the alveolar lumen could morphologically be identified as neutrophils (-3a- and -3b-), while these immune cells were still absent at that time post-IMI with <i>S. aureus</i> (-4-) or in sham-inoculated (PBS, -11-) and lactating (at 0 h,-12-) glands. In contrast, the influx of neutrophils could only be detected at 24 h post-IMI with <i>S. aureus</i> (-8-). A clear nuclear translocation of NF-kappaB p65 could be detected at 12 h post-IMI with <i>E. coli</i> and 24 h post-IMI with <i>S. aureus</i> (-3a- and -8a-) in these immune cells. At 24 h post-IMI with <i>E. coli</i>, epithelial damage at mammary alveoli was characterized by budding of mammary epithelia (-5b-) and resulted in shedded epithelial cells (-5a-) and empty spots in the luminal layer (-5c-), while at 24 h post-IMI with <i>S. aureus</i> only some cells started to protrude from the epithelial layer (-10b-). (B) IL-8 like chemokines (i.e. KC and MIP-2) confirmed this difference in kinetics of the neutrophil influx between both pathogens. Both chemokines were significantly induced in <i>E. coli</i> infected glands compared to sham-inoculated glands (PBS) already at 12 h post-IMI (a), while for <i>S. aureus</i> this strong induction was only seen at 24 h post-IMI (b). Letters of homogeneous subsets were marked if the difference between treatments were statistically significant (P<0.05)</p

Differential mammary IL-1beta fragmentation post-IMI with <i>E. coli</i> versus <i>S. aureus</i> and effect on bacterial growth.

<p>(A) Cleavage of IL-1beta in the mammary gland post-IMI with <i>E. coli</i> shows a fast, transient IL-1beta maturation with six IL-1beta fragments at 12h i.e. ±30 kDa, 25 kDa, ±20 kDa, ±17 kDa, ±15 kDa and ±10 kDa (fragments 1, 2, 3, p17, 4 and 5, respectively). At 24 h (only fragment 1 and p31 proform) is seen. In IL-1beta KO and in sham-inoculated (PBS) mice no fragments or p31 proform are detected. Despite clear procaspase-1 maturation, the early complex IL-1beta pattern is not the result of caspase-1 cleavage as the latter only occurs extensively at 24 h and as cleavage of pro-IL-1beta still occurs in caspase-1 KO glands (right Western blot images). No caspase-1 maturation was detected in caspase-1 KO or in sham-inoculated mammary glands. (B) Cleavage of IL-1beta in the mammary gland post-IMI with <i>S. aureus</i> shows a slower IL-1beta maturation with four IL-1beta fragments at 24 h i.e. ±30 kDa, ±20 kDa, ±18 kDa and ±17 kDa (fragments 1, 3, 6 and p17, respectively). At 12 h (only fragment 1 i.e the preform p31, 2, 3,and p17). The late IL-1beta maturation is not the result of caspase-1 cleavage as IL-1beta cleavage still occurs in caspase-1 KO glands (strong p17 fragment albeit in the absence of the p31 proform). Both procaspase-1 and its cleavage were low, respectively absent in IL-1beta KO glands (right Western blot images). No IL-1beta or caspase-1 was detected in sham-inoculated mammary glands of wT mice. (C) ProIL-1beta fragmentation affects bacterial growth as in IL-1beta KO mammary glands on time points of interest (12 h for <i>E. coli</i>, 24 h for <i>S. aureus</i>) CFU counts are significantly higher for both pathogens than in wT mammary glands, especially for <i>E. coli</i> (P<0.01 *, P<0.001**). DL = detection limit.</p

CNF1-triggered immunity requires inflammatory caspases.

<p>(A and B) IL-1β production and maturation/secretion after treatment with CNF1, LPS or CNF1 (1 μg/ml) + LPS (100 ng/ml) for 10 h. Actin and BSA were used as loading controls. (B) Graph showing the quantification of IL-1β secretion normalized to the control (n = 3). (C and D) Female C57BL/6 WT (C) or congenic C1^-/-C11^-/- mice (D) were intravenously infected with 10⁷ CFUs of <i>E</i>. <i>coli</i>^CNF1+ or with the isogenic CNF1-deleted mutant (<i>E</i>. <i>coli</i>^CNF1-) prior to the collection of peripheral blood at 3, 6, 24 and 48 h for bacteremia measurements. The data are expressed as the mean ± SEM (n = 7–8). (E and F) Analysis of the circulating levels of IL-1β (E) and KC (F) in the mouse sera by ELISA. Serum samples from mice infected with 10⁷<i>E</i>. <i>coli</i>^CNF1+ or with the isogenic mutant <i>E</i>. <i>coli</i>^CNF1- were collected at 3 h after intravenous infection and analyzed by ELISA (n = 3). P-values<0.05 (*); and P-values<0.01 (**) were considered statistically significant.</p

CNF1-triggered IL-1β maturation requires activated Rac, ASC and caspase-1.

<p>(A) Western blot analysis of the production and maturation/secretion of IL-1β by primary macrophages following treatment with CNF1, LPS or CNF1+LPS for 10 h. Actin and BSA were used as loading controls. (B) Quantification of caspase-1 activity in macrophages following treatment with CNF1+LPS for 6 h using YVAD-Fluorescent Labelled Inhibitor Caspase-1 Activity (FLICA). (C) Western blot analysis of macrophages IL-1β maturation/secretion upon transfection of HA-Rac2^Q61E and LPS treatment. (D) Co-immunoprecipitation of Myc-Rac2 and caspase-1 using an anti-Myc antibody following the treatment of HEK 293T cells with CNF1+LPS for 6 h.</p

CNF1 potentiates LPS-triggered immune responses.

<p>(A) Monocytes (5x10⁵ cells per condition) isolated from mouse blood were treated with PBS (control) or with 1 μg/ml CNF1 toxin for 10 h with or without 100 ng/ml LPS. Cell culture supernatants were analyzed using mouse ELISArray kits (n = 3). The data are shown as fold inductions compared with the control condition. (B, C and D) Monocytes (5x10⁵ cells per condition) purified from mouse blood were treated for 10 h. Cells were treated as indicated with 0.1, 1, or 10 μg/ml of CNF1 toxin or the CNF1 mutant C866S alone or in combination with ultrapure <i>E</i>. <i>coli</i> LPS at 1, 10 or 100 ng/ml (n = 3). (B) KC, (C) IL-6, and (D) IL-1β cytokine secretion was analyzed using ELISA (n = 4). (E) Female BALB/c mice were intravenously infected with 10⁷ CFUs of <i>E</i>. <i>coli</i> expressing CNF1 (<i>E</i>. <i>coli</i>^CNF1+) + PBS as a control or with an <i>E</i>. <i>coli</i>^CNF1+ + IL-1β antagonist (Kineret; 1.5 mg/kg) prior to the collection of peripheral blood at 3, 6, 24 and 48 h for the measurement of bacteremia (n = 10). P-values <0.05 (*); and P-values <0.01 (**) were considered statistically significant.</p