(A) Activation of ERK in C3H/HeOuJ and C3H/HeJ peritoneal macrophages in response to LPS treatment or infection with wild-type S. enterica serovar Typhimurium. Macrophages were treated with 100 ng of LPS per ml or were infected with 10⁷ SL1344 bacteria for the times indicated. Cell lysates were prepared and analyzed by Western blotting for the presence of phospho-ERK (p-ERK). The blots were then stripped and reprobed to detect total ERK. (B) Quantitation of the Western blot in panel A. The autoradiograph was scanned and analyzed by using NIH Image software. The mean pixel densities of the phospho-ERK bands (normalized to the pixel densities of the corresponding total ERK bands) are shown. (C) Degradation of IκBα in C3H/HeOuJ and C3H/HeJ peritoneal macrophages in response to infection with wild-type S. enterica serovar Typhimurium. Macrophages were infected with 10⁷ SL1344 bacteria for the times indicated. Cell lysates were prepared and analyzed by Western blotting for the presence of IκBα. The blot was then stripped and reprobed for the presence of actin to confirm equivalent loading of lanes. Quantitation of the Western blot is shown at the bottom, and the data indicate the mean pixel densities of the IκBα bands (normalized to the pixel densities of the corresponding actin bands) expressed as percentages of the value at time zero for each strain. (D) Activation of ERK in C3H/HeJ peritoneal macrophages in response to S. enterica serovar Typhimurium or its products. Macrophages were left untreated (C) or were exposed to 10⁷ wild-type S. enterica serovar Typhimurium strain SL1344 bacteria (S), 10⁷ heat-killed SL1344 bacteria (Hk), a 5% (vol/vol) supernatant of a stationary culture of wild-type S. enterica serovar Typhimurium (Sup), a 5% (vol/vol) supernatant of a stationary culture of flagellin-deficient S. enterica serovar Typhimurium (F-), 5% (vol/vol) LB broth (LB), or 100 ng of LPS per ml (L) for 45 min. Cell lysates were prepared and analyzed by Western blotting for the presence of phospho-ERK. The blots were then stripped and reprobed to detect total ERK. Quantitation of the Western blot is shown at the bottom, and the data indicate the mean pixel densities of the phospho-ERK bands normalized to the pixel densities of the corresponding total ERK bands.

(A) Pretreatment of C3H/HeJ peritoneal macrophages with S. enterica serovar Typhimurium products results in tolerization of ERK activation by PG. Macrophages were not pretreated (−) or were pretreated overnight with 10⁷ heat-killed wild-type S. enterica serovar Typhimurium SL1344 bacteria (Hk), 5% (vol/vol) culture supernatant of SL1344 (Sup), 5% (vol/vol) LB broth (LB), 100 ng of LPS per ml (L), or 25 μg of PG per ml (P). They were then left unstimulated (C) or were stimulated for 45 min with 10⁷ live wild-type SL1344 bacteria (S), 25 μg of PG per ml (P), or 1 μg of phorbol myristate acetate per ml (Pm). Cell lysates were prepared and analyzed by Western blotting for the presence of phospho-ERK (p-ERK). Pre-Tx, pretreatment; Tx, treatment. (B) Quantitation of the Western blots shown in panel A. The autoradiographs were scanned and analyzed by using NIH Image software. The mean pixel densities of the phospho-ERK bands (normalized to the pixel densities of the corresponding total ERK bands) are shown. (C) Effect of pretreatment of peritoneal macrophages with heat-killed S. enterica serovar Typhimurium on subsequent TNF-α production in response to PG. Macrophages were not pretreated or were pretreated overnight with 25 μg of PG per ml or with 10⁷ heat-killed wild-type S. enterica serovar Typhimurium SL1344 bacteria. After the pretreatment stimulus was washed away, all groups of macrophages were stimulated for 2 h with 25 μg of PG per ml, and the supernatants were analyzed by ELISA for TNF-α. The normalized amount of TNF-α was expressed as a percentage of the amount produced by the nonpretreated, PG-stimulated cells. The data are the means of the results of three separate experiments, and the error bars indicate standard deviations. The P values for the comparisons are <0.0005 (one asterisk) and <0.0005 (two asterisks).

Model for the role of TLR4 and other PRRs in the Salmonella-macrophage interaction. Salmonella activates proximal signaling pathways via TLR4 and other PRRs. TLR4 signals are essential at a posttranscriptional step for high-level expression of secreted TNF-α protein. Signals provided by other PRRs are sufficient for induction of the TNF-α transcript and at least some genes associated with tolerance but not for production of high levels of secreted TNF-α. The dashed arrow indicates the signal required for high-level secreted TNF-α production that PRRs other than TLR4 are ineffective at providing.

Production of TNF-α by peritoneal macrophages from C3H/HeOuJ and C3H/HeJ mice. Macrophages were not stimulated (C), were stimulated with 100 ng of LPS per ml (L) or 25 μg of PG per ml (P), or were infected with 10⁷ wild-type S. enterica serovar Typhimurium strain SL1344 (S) for 2 h, and the supernatants were analyzed by ELISA for TNF-α. The amount of TNF-α produced was normalized to the protein content of the macrophage cell lysate. The data are the means of the results of at least four separate experiments, and the error bars indicate standard deviations. The P values for the differences between the responses of the two strains to LPS, Salmonella, and PG were <0.0005 (one asterisk), <0.001 (two asterisks), and <0.0025 (three asterisks), respectively.

(A) Induction of TNF-α mRNA in C3H/HeOuJ and C3H/HeJ macrophages by Salmonella infection: semiquantitative RT-PCR analysis. Macrophages were not infected (lanes C) or were infected for 2 h with 10⁷ wild-type S. enterica serovar Typhimurium SL1344 bacteria (lanes S). Total RNA was prepared and used for the RT-PCR with primers specific for either TNF-α or the housekeeping transcript GAPDH. Aliquots of the PCR products were analyzed by agarose gel electrophoresis and were visualized by UV transillumination. (B) Induction of TNF-α mRNA in C3H/HeOuJ and C3H/HeJ macrophages by Salmonella infection: quantitative real-time PCR analysis. Macrophages were not infected (bars C) or were infected for 2 h with 10⁷ wild-type S. enterica serovar Typhimurium SL1344 bacteria (bars S). Total RNA was prepared, reverse transcribed, and amplified with an iCycler by using primers specific for either TNF-α or GAPDH in the presence of SYBR Green. Normalized C_t values were derived as described in Materials and Methods and are expressed as the differences from control, uninfected cells.

Induction of genes associated with macrophage tolerance by Salmonella. Peritoneal macrophages from either C3H/HeOuJ or C3H/HeJ mice were left untreated (C) or were treated overnight with 100 ng of LPS per ml (L) or 10⁷ heat-killed wild-type S. enterica serovar Typhimurium SL1344 bacteria (S). Protein lysates were prepared the next morning and analyzed by Western blotting with antibodies to NF-κB p50, NF-κB p52, or actin. The arrows indicate the relevant bands. Quantitation of the Western blots is shown at the bottom, and the data indicate the mean pixel densities of the p50 or p52 bands normalized to the pixel densities of the corresponding actin bands.