Differential Recognition of Vibrio parahaemolyticus OmpU by Toll-Like Receptors in Monocytes and Macrophages for the Induction of Proinflammatory Responses.

Vibrio parahaemolyticus is a human pathogen, and it is a major cause of severe gastroenteritis in coastal areas. OmpU is one of the major outer membrane porins of V. parahaemolyticus Host-immunomodulatory effects of V. parahaemolyticus OmpU (VpOmpU) have not been elucidated yet. In this study, in an effort towards characterizing the effect of VpOmpU on innate immune responses of the host, we observed that VpOmpU is recognized by the Toll-like receptor 1/2 (TLR1/2) heterodimer in THP-1 monocytes but by both TLR1/2 and TLR2/6 heterodimers in RAW 264.7 macrophages. To the best of our knowledge, this is the first report of a natural pathogen-associated molecular pattern (PAMP) recognized by both TLR1/2 and TLR2/6 heterodimers; so far, mainly the synthetic ligand Pam2CSK4 has been known to be recognized by both the TLR1/2 and TLR2/6 heterodimers. We also have shown that VpOmpU can activate monocytes and macrophages, leading to the generation of proinflammatory responses as indicated by tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO production in macrophages and TNF-α and IL-6 production in monocytes. VpOmpU-mediated proinflammatory responses involve MyD88-IRAK-1 leading to the activation of mitogen-activated protein (MAP) kinases (p38 and Jun N-terminal protein kinase [JNK]) and transcription factors NF-κB and AP-1. Further, we have shown that for the activation of macrophages leading to the proinflammatory responses, the TLR2/6 heterodimer is preferred over the TLR1/2 heterodimer. We have also shown that MAP kinase activation is TLR2 mediated.

Gulati A ，Kumar R ，Mukhopadhaya A 《-》

Vibrio cholerae porin OmpU mediates M1-polarization of macrophages/monocytes via TLR1/TLR2 activation.

Polarization of the monocytes and macrophages toward the M1 and M2 states is important for hosts' defense against the pathogens. Moreover, it plays a crucial role to resolve the overwhelming inflammatory responses that can be harmful to the host. Polarization of macrophages/monocytes can be induced by pathogen-associated molecular patterns (PAMPs). PAMP-mediated monocyte/macrophage polarization is important during the infection, as pathogen can suppress host immune system by altering the polarization status of the macrophages/monocytes. OmpU, an outer membrane porin protein of Vibrio cholerae, possesses the ability to induce pro-inflammatory responses in monocytes/macrophages. It is also able to down-regulate the LPS-mediated activation of the monocytes/macrophages. Such observation leads us to believe that OmpU may induce a state that can be called as M1/M2-intermediate state. In the present study, we evaluated a set of M1 and M2 markers in RAW 264.7 murine macrophage cell line, and THP-1 human monocytic cell line, in response to the purified OmpU protein. We observed that OmpU, as a PAMP, induced M1-polarization by activating the Toll-like receptor (TLR) signaling pathway. OmpU induced formation of TLR1/TLR2-heterodimers. OmpU-mediated TLR-activation led to the MyD88 recruitment to the TLR1/TLR2 complex. MyD88, in turn, recruited IRAK1. Ultimately, OmpU-mediated signaling led to the activation and subsequent nuclear translocation of the NFκB p65 subunit. We also observed that blocking of the TLR1, TLR2, IRAK1, and NFκB affected OmpU-mediated production of M1-associated pro-inflammatory cytokines such as TNFα and IL-6.

Khan J ，Sharma PK ，Mukhopadhaya A 《-》

Transmembrane oligomeric form of Vibrio cholerae cytolysin triggers TLR2/TLR6-dependent proinflammatory responses in monocytes and macrophages.

Khilwani B ，Mukhopadhaya A ，Chattopadhyay K 《-》

Vibrio cholerae Porin OmpU Activates Dendritic Cells via TLR2 and the NLRP3 Inflammasome.

Dhar V ，Gandhi S ，Sakharwade SC ，Chawla A ，Mukhopadhaya A ... -  《-》

Vibrio cholerae OmpU Mediates CD36-Dependent Reactive Oxygen Species Generation Triggering an Additional Pathway of MAPK Activation in Macrophages.

OmpU, one of the porins of Gram-negative bacteria Vibrio cholerae, induces TLR1/2-MyD88-NF-κB-dependent proinflammatory cytokine production by monocytes and macrophages of human and mouse origin. In this study, we report that in both the cell types, OmpU-induced proinflammatory responses involve activation of MAPKs (p38 and JNK). Interestingly, we observed that in OmpU-treated macrophages, p38 activation is TLR2 dependent, but JNK activation happens through a separate pathway involving reactive oxygen species (ROS) generation by NADPH oxidase complex and mitochondrial ROS. Further, we observed that OmpU-mediated mitochondrial ROS generation probably depends on OmpU translocation to mitochondria and NADPH oxidase-mediated ROS production is due to activation of scavenger receptor CD36. For the first time, to our knowledge, we are reporting that a Gram-negative bacterial protein can activate CD36 as a pattern recognition receptor. Additionally, we found that in OmpU-treated monocytes, both JNK and p38 activation is linked to the TLR2 activation only. Therefore, the ability of macrophages to employ multiple receptors such as TLR2 and CD36 to recognize a single ligand, as in this case OmpU, probably explains the very basic nature of macrophages being more proinflammatory than monocytes.

Prasad GVRK ，Dhar V ，Mukhopadhaya A 《-》