TLR4, MyD88, and MyD88-dependent and -independent pathways are necessary for Hp91-mediated activation of antigen presenting cells. (A) BM-DCs from wild type (WT) or knockout mice were exposed to 200 μg/ml Hp91. Supernatants were analyzed for IL-6 by ELISA. Results are mean (±SEM) for N = 3-5. (B) J774 macrophages were stimulated for indicated times with 200 μg/ml Hp91, 10 ng/ml LPS or left untreated (M). Lysates were analyzed for p-p38 by WB. Blots were probed with anti-p-p38, anti-p38, and anti-GAPDH antibodies. One of N=4. (C) J774 macrophages were pretreated with media (Med), DMSO control (D), Dynasore (DYN) 80 μM, phenylarsine oxide (PAO) 2 μM or chlorpromazine (CP) 100 μM before incubation with 200 μg/ml Cp488-labeled Hp91 for 30 min. Cells were analyzed by flow cytometry. Results are mean (±SEM), N=4. (D) J774 macrophages were pretreated with DMSO control (D), SB203580 (SB) 20 μM, PD98059 (PD) 20 μM, TPCK 20 μM, Dynasore (DYN) 80 μM, phenylarsine oxide (PAO) 2 μM, or chlorpromazine (CP) 100 μM then exposed to Hp91. Supernatants were analyzed for IL-6 by ELISA. Data are mean (±SEM), N=3. (E) RAW 264.7 macrophages were pretreated with medium, DMSO control, or Dynasore (DYN) 80 μM prior to stimulation for indicated times with Hp91 (200 μg/ml), LPS (10 ng/ml) or media. Cell lysates were analyzed for p-IRF3. Immunoblots were probed with anti-p-IRF3, anti-IRF3, and anti-β-actin antibodies. (F) cDNA from stimulated J774 cells were evaluated for IFN-α2 mRNA by qPCR. Values are normalized against endogenous GAPDH controls, in duplicate. (G) Wildtype or MyD88-/- knockout mice were immunized with SIINFEKL peptide in PBS with or without Hp91. Splenocytes from immunized mice were cultured with SIINFEKL peptide (2.5 μg/ml). The number of IFN-γ-secreting cells was determined 18 h later. Data are mean (±SEM), 5–10 mice/group. *p < 0.05; Student’s t-test.