Additional file 5: Figure S4. of Urinary chitinase 3-like protein 1 for early diagnosis of acute kidney injury: a prospective cohort study in adult critically ill patients

Distribution of urinary chitinase 3-like protein 1 (UCHI3L1) (a) and urinary neutrophil gelatinase-associated lipocalin (UNGAL) (b) at enrollment in the eight selected subgroups of patients who did not develop acute kidney injury (AKI) based on the Kidney Disease | Improving Global Outcomes serum creatinine (SCr) criteria (No AKI SCr ) within 7 days after enrollment, compared to the distribution in all patients with no AKISCr within 24 h after enrollment, and in all those maximally reaching AKISCr stages 1, 2, or 3 within 24 h after enrollment. (TIF 716 kb

Additional file 2: Figure S1. of Urinary chitinase 3-like protein 1 for early diagnosis of acute kidney injury: a prospective cohort study in adult critically ill patients

Area under the receiver-operating characteristics curve (AUC-ROC) with 95 % confidence interval (CI) of urinary chitinase 3-like protein 1 (UCHI3L1) (a) and urinary neutrophil gelatinase-associated lipocalin (UNGAL) (b) at enrollment for predicting acute kidney injury (AKI) stage ≥2 based on the Kidney Disease | Improving Global Outcomes (KDIGO) serum creatinine (SCr) or urine output (UO) criteria (AKI SCr/UO ) within 12 h in different subgroups of patients. (TIF 434 kb

Additional file 4: Figure S3. of Urinary chitinase 3-like protein 1 for early diagnosis of acute kidney injury: a prospective cohort study in adult critically ill patients

Distribution of urinary chitinase 3-like protein 1 (UCHI3L1) (a) and urinary neutrophil gelatinase-associated lipocalin (UNGAL) (b) at enrollment in the eight selected subgroups of patients who did not develop acute kidney injury (AKI) based on the Kidney Disease | Improving Global Outcomes serum creatinine (SCr) or urine output (UO) criteria (No AKI SCr/UO ) within 7 days after enrollment, compared to the distribution in all patients with no AKISCr/UO within 12 h after enrollment, and in all those maximally reaching AKISCr/UO stages 1, 2, or 3 within 12 h after enrollment. (TIF 715 kb

image_1_Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.tif

<p>Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders’ innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1^−/− mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.</p

image_2_Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.tif

<p>Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders’ innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1^−/− mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.</p

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

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

<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