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Pattern recognition receptors (PRRs) have the ability to recognize conserved molecular patterns on pathogens and initiate an innate immune response. However, in vivo studies have shown that PRRs, such as receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs), have an antagonistic effect in Mycobacterium tuberculosis infections. Therefore, we investigated whether RAGE and TLR2 have a synergistic effect on cytokine production by mouse peritoneal macrophages stimulated with Mycobacterium smegmatis. We found that peritoneal macrophages from mice treated with anti-TLR2 had a lower TNF-α production than untreated mice. However, mice treated with anti-RAGE had a higher TNF-α production than untreated mice. Similarly, anti-TLR2 and anti-RAGE treatments in combination resulted in lower TNF-α production than observed in the anti-TLR2 treatment. In addition, when macrophages were treated with anti-TLR2 and anti-RAGE simultaneously, they produced lower levels of TNF-α than with anti-TLR2 treatment alone. Mycobacterial DNA was detected by a multiplex polymerase chain reaction method in macrophages treated with anti-TLR2 and anti-RAGE simultaneously, and this was confirmed by the addition of anti-TLR2 and anti-RAGE to the culture medium for 4 days. TLR2 and RAGE protein were found to co-localize on the surface of the macrophages by immunofluorescence analysis. These findings showed that 0b46394aab