<i>B</i>. <i>infantis</i>-induced IL-1ß secretion by THP-1 macrophages involves caspase 1.

<p><b>A.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> or <i>C</i>. <i>rodentium</i> for 1 hour, washed, then incubated in fresh medium for 4 hours. ZYVAD at a final concentration of 50 or 100 μM was present in the medium, as indicated, throughout the course of the experiment. Supernatants were collected at the end of the 4 hour incubation and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, **<i>p</i> = 0.0002, n = 6 per experimental condition. <b>B.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> for 1 hour, washed, then incubated in fresh medium for the times indicated before preparation of cell lysates. The lysates were analyzed by western blotting with an antibody to caspase 1, with equal loading of lanes being confirmed by blotting with an anti-ß-actin antibody. The p10 processed form of caspase 1 is shown.</p

<i>B</i>. <i>infantis</i> and <i>B</i>. <i>fragilis</i> induce IL-1ß secretion in immortalized mouse macrophages in an NLRP3- and potassium efflux-dependent fashion.

<p><b>A.</b> Immortalized WT mouse macrophages were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> (as well as a 5-fold higher number (5X) in the case of heat-killed <i>B</i>. <i>infantis</i>) for 1 hour in the presence or absence of 5 μM cytochalasin D (CytD) as indicated. The cells were washed and incubated overnight in fresh medium, with cytochalasin D added back as appropriate. IL-1ß concentrations in the supernatants were determined by ELISA. In the condition with 5X heat-killed bacteria, the cells were exposed continuously to the bacteria overnight without the intervening wash step. *<i>p</i> = 0.0001, **<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> WT or NLRP3 KO immortalized mouse macrophages were infected with live <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> for 1 hour. The cells were washed and incubated overnight in fresh medium. IL-1ß concentrations in the supernatants were determined by ELISA. *<i>p</i> < 0.0001, **<i>p</i> = 0.0001, n = 6 per experimental condition. <b>C.</b> Immortalized WT mouse macrophages were infected with live <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> for 1 hour in the presence or absence of 50 mM potassium chloride (KCl) or 2 μM ruthenium red (RR) as indicated. The cells were washed and incubated overnight in fresh medium, with potassium chloride or ruthenium red added back as appropriate. IL-1ß concentrations in the supernatants were determined by ELISA. *<i>p</i> = 0.0002, **<i>p</i> = 0.0011, ***<i>p</i> < 0.0001, n = 6 per experimental condition.</p

<i>B</i>. <i>infantis</i> and <i>B</i>. <i>fragilis</i> induce IL-1ß secretion in mouse BMDMs after overnight incubation.

<p><b>A.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i>, <i>B</i>. <i>fragilis</i> or <i>C</i>. <i>rodentium</i> for 1 hour. The cells were washed and incubated overnight in fresh medium. IL-1ß concentrations in the supernatants were determined by ELISA. *<i>p</i> = 0.0003, **<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i>, <i>B</i>. <i>fragilis</i> or <i>C</i>. <i>rodentium</i> as in <b>A</b> and then incubated overnight (upper panel) or for the times indicated (6 hours or overnight–O/N–lower panel). Cell lysates and supernatants were immunoblotted to detect pro-IL-1ß and mature IL-1ß, respectively, while cell lysates were blotted with an anti-actin antibody to confirm equal loading of lanes. The caspase inhibitor ZYVAD was added to the cells at a concentration of 100 μM where indicated. <b>C.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> for 1 hour. The cells were washed, placed in fresh medium and incubated for a further 4 hours. Total cellular RNA was prepared and used to determine relative IL-1ß mRNA expression by qRT-PCR. *<i>p</i> = 0.0003, **<i>p</i> < 0.0001, n = 6 per experimental condition. <b>D.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i> for 1 hour, washed, then incubated in fresh medium for 4 hours in the presence or absence of 5 mM ATP added exogenously during the last 1 hour. Supernatants were collected at the end of the 4 hour incubation and were analyzed by ELISA to determine IL-1ß concentrations. *<i>p</i> < 0.0001, n = 6 per experimental condition.</p

Bacterial viability and phagocytosis are not required for <i>B</i>. <i>infantis</i>- and <i>B</i>. <i>fragilis</i>-induced IL-1ß secretion by mouse BMDMs.

<p><b>A.</b> Mouse BMDMs were infected with live <i>B</i>. <i>infantis</i> or heat-killed (HK) bacteria, either equivalent in number to the live organisms or a 5-fold excess (5X), for 1 hour. The cells were washed and incubated overnight in fresh medium. IL-1ß concentrations in the supernatants were determined by ELISA. In the condition with 5X heat-killed bacteria, the cells were exposed continuously to the bacteria overnight without the intervening wash. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> Mouse BMDMs were infected with live <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> or an equivalent number of heat-killed (HK) <i>B</i>. <i>fragilis</i> for 1 hour in the presence or absence of 5 μM cytochalasin D (CytD, dissolved in DMSO) as indicated. The cells were washed and incubated overnight in fresh medium, with cytochalasin D added back as appropriate. IL-1ß concentrations in the supernatants were determined by ELISA. n = 6 per experimental condition.</p

<i>B</i>. <i>infantis</i>- and <i>B</i>. <i>fragilis</i>-induced IL-1ß secretion by THP-1 macrophages is dependent on potassium efflux but does not require bacterial viability or phagocytosis.

<p><b>A.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> for 1 hour in the presence or absence of 50 mM potassium chloride (KCl) or 2 μM ruthenium red (RR), as indicated. The cells were washed and incubated in fresh medium, with potassium chloride or ruthenium red added back as appropriate. Supernatants were collected at the end of 4 hours and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> for 1 hour. The cells were washed and incubated in fresh medium, with 5 units/ml of apyrase added as indicated. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA, n = 6 per experimental condition. <b>C.</b> THP-1 macrophages were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>infantis</i> for 1 hour, in the presence of 5 μM cytochalasin D (CytD) where indicated. The cells were washed and incubated in fresh medium, with cytochalasin D added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA, n = 6 per experimental condition. <b>D.</b> THP-1 macrophages were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>fragilis</i> for 1 hour, in the presence of 5 μM cytochalasin D (CytD) or 50 mM potassium chloride (KCl) where indicated. The cells were washed and incubated in fresh medium, with cytochalasin D, potassium chloride or 5 units/ml of apyrase added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA. *p < 0.0001, n = 6 per experimental condition.</p

<i>B</i>. <i>infantis</i>-induced IL-1ß secretion by primary human MDMs.

<p><b>A.</b> Primary human MDMs were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>infantis</i> for 1 hour in the presence or absence 5 μM cytochalasin D (Cyt D) or 50 mM potassium chloride (KCl) as indicated. The cells were washed, then incubated in fresh medium, with potassium chloride added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> Primary human MDMs were infected with heat-killed <i>B</i>. <i>infantis</i> for 1 hour in the presence or absence 5 μM cytochalasin D (Cyt D) or 50 mM potassium chloride (KCl) as indicated. The cells were washed, then incubated in fresh medium, with potassium chloride added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>C.</b> Primary human MDMs were treated with 10 μg/ml of Pam3Cys for 4 hours, after which cellular RNA was analyzed by quantitative RT-PCR to determine IL-1ß mRNA levels (left panel) and supernatants were analyzed by ELISA to determine secreted IL-1ß concentrations (right panel). *<i>p</i> = 0.0002, n = 6 per experimental condition.</p

Effects of TNFα on hepcidin expression in Huh7 cells.

<p><b>A</b>, Huh7 cells were treated with the indicated concentrations of TNFα for 12 hours and hepcidin expression, normalized to actin, was determined relative to the mean of untreated cells. *p<0.0001, n = 5 for each condition. <b>B</b>, Huh7 cells were treated for 12 hours with 10 ng/ml of IL-6, either alone or together with 10 ng/ml of TNFα, and hepcidin expression, normalized to actin, was determined relative to the mean of control, untreated cells. *p = 0.014, n = 3 for each condition. <b>C</b>, Huh7 cells were transfected with a firefly luciferase reporter under the control of the hepcidin promoter along with a constitutively expressed Renilla reporter. Forty-eight hours after transfection, the cells were treated with 10 ng/ml of TNFα for 12 hours and luciferase activities determined. The ratio of firefly to Renilla (F/R) luciferase activity is indicated. *p = 0.002, n = 3 for each condition.</p

Effects of recombinant TNFα on the BMP/Smad pathway in non-colitic mice.

<p><b>A</b>, Immunoblotting of liver lysates for total Smad1 and GAPDH in non-colitic mice treated with PBS or 50 µg/kg body weight recombinant TNFα (rTNFα) followed by sacrifice 16 hours later. Each lane represents an individual mouse. The lanes with the lysates from the rTNFα-treated mice have been over-loaded to clearly illustrate the decrease in Smad1 levels. <b>B</b>, Quantitation of band intensities from immunoblotting. *p = 0.0001, **p = 0.0004, ***p = 0.01, n = 3 mice per group. <b>C</b>, Liver Smad1 mRNA levels in the non-colitic mice treated with PBS or rTNFα, shown relative to the mean of PBS-treated mice after normalizing to GAPDH. n = 8 (control), 6 (rTNFα).</p

Effect of TNFα on hepcidin expression in vivo.

<p>Mice without colitis were treated with PBS or recombinant TNFα (rTNFα) for 6 or 16 hours as described in the text. Liver hepcidin expression was analyzed and is shown relative to the mean of PBS-treated mice after normalizing to GAPDH. *p = 0.005, n = 8 (PBS), 6 (each of the TNFα treated groups).</p

Alterations in the BMP/Smad pathway in DSS colitis.

<p><b>A</b>, Representative immunoblotting of liver lysates for phosphorylated Smad (pSmad) 1/5/8, total Smad1 and actin in control untreated mice (Con), or mice treated for 3 or 7 days with DSS. Each lane represents an individual animal. <b>B</b>, Quantitation of band intensities from the immunoblotting experiment. *p = 0.001, **p = 0.001, n = 6 mice per group.</p