Research in the Immunopathology Section focuses on the biological mediators and signal transduction pathways involved in the modulation of human monocyte and lymphocyte functions that may contribute to the immunopathology associated with various inflammatory lesions. Monocytes/macrophages are prominent in many inflammatory diseases, such as periodontal disease, rheumatiod arthritis, atherosclerosis, and cancer. The pathology associated with these diseases involves alterations in the integrity of the connective tissue framework implicating a role for matrix metalloproteinases (MMPs). MMPs are comprised of a family of zinc dependent endopeptidases divided into major subgroups that include the interstitial collagenases, the gelatinases, stromelysins, membrane type MMPs and others. Collectively these enzymes are capable of degrading all the extracellular matrix components. Because MMPs and tissue inhibitors of MMPs (TIMPs) are believed to play a major role in the destruction and remodeling of connective tissue, a major emphasis has been placed on how these enzymes and inhibitors are regulated in the human monocyte/macrophage as well as in the reciprocal interaction between monocytes/macrophages and tumor cells. Interaction between monocytes/macrophages and human oral squamous carcinoma cells (HNSCC) in the production and activation of MMPs The stromal cells surrounding cancer sites are thought to play a major role in the degree of tumor progression. A significant portion of these stromal cells are comprised of monocytes and macrophages that have been polarized by the specific cytokines present at the site into subtypes known as M1 and M2 macrophages. The MMPs produced by these subtypes of macrophages may be important determinates in the extent of cancer metastasis through the digestion of specific extracellular matrix surrounding the cancer site. Our previous qPCR studies have identified the differential expression levels of the various members of the MMP family and TIMPs by monocytes, macrophages and their subtypes in the presence or absence of specific cytokines and endotoxin. In addition to the levels of MMPs produced by stromal cells, HNSCCs also produce MMPs and TIMPs the levels of which tend to vary depending on the specific HNSCCs. To examine the base line levels and variability of MMPs and TIMPs of HNSCCs we examined five HNSCC lines. We found considerable differences in the expression profile between these HNSCC lines. For example NH12 and OSCC3 cell lines express very low to undetectable levels of MMP-1, -2, -3, -10 and -11 compared to HN30, Cal27 and HN4 cell lines which express high levels of these MMPs. Our next step in this study is to determine the effect of co-cultures of human oral squamous carcinoma cells (HNSCC) on the expression MMPs and TIMPs by monocytes and macrophages. HNSCCs and monocytes/macrophages are being cultured in wells containing inserts to separate the populations. Expression levels of MMPs and TIMPs in the individual populations are being determined by qPCR. We are addressing the following questions in this study. Do HNSCCs instruct stromal cells to express MMPs and TIMPs? Are those HNSCC lines that produce low levels of specific MMPs compensating by inducing the production of those MMPs by monocytes/macrophages? Conversely, do monocytes/macrophages induce HNSCCs to produce MMPs that are normally expressed at low or non-detectable levels? Are monocyte/macrophages involved in the activation of pro-MMPs produced by HNSCCs? Regulation of monocyte/macrophage MMP production by inhibitors used to target signaling pathways in tumor cells. The PI3K/AKT/mTOR pathway is major signal transduction pathway involved in tumor progression and considerable effort has been aimed at designing pharmacological inhibitors to interrupt this pathway. In addition to modulating tumor cell signaling these agents also have a potential impact on signaling in stromal cells and their regulation of connective tissue turnover. Our recent studies have targeted the role of AKT in the regulation of MMP production by monocytes. Stimulation of monocytes with LPS results in the activation of AKT as demonstrated by the phosphorylation of Ser 473 and Thr 308. We have shown that monocytes in which AKT has been inhibited have enhanced expression and production of MMP-1. This is in contrast to previous findings in which we demonstrated that MMP-9 production was inhibited when AKT activation was blocked. Examination of the mechanism involved in enhancement of monocyte MMP-1 production as a result of inhibition of AKT revealed that it is unrelated to downstream activation of mTOR since rapamycin failed in increase MMP-1 in activated monocytes. However, inhibition of AKT resulted in an increase in the activation of p38 MAPK and a decrease in ERK1/2 MAPK phosphorylation. Based on previous findings from our laboratory this may explain, in part, the differential regulation of MMP-1 and -9. We are currently focusing on glycogen synthase kinase -3 (GSK-3&#946;), a downstream target of AKT that is inactivated when phosphorylated (pS9GSK-3&#946;) by AKT. pS9GSK-3&#946;is known to exert both pro- and anti-inflammatory effects through its interaction with transcription factors. We are currently examining the role of GSK-3&#946;in differential regulation of MMPs through the use of specific inhibitors. Involvement of TLR2 and TLR4 in cell responses to Rickettsia akari In an ongoing collaboration with investigators at the University of Maryland we have been examining the role of TLRs in endotoxin signaling which leads to endotoxin tolerance. TLRs are primary sensors of microbial pathogens that activate innate immune cells, as well as initiate and orchestrate adaptive immune responses. Our recent studies have focused on the role of TLR2 and TLR4 in the recognition of Rickettsia akari, a causative agent of rickettsialpox. The findings in this paper demonstrate that TLR2 and TLR4 are innate immune sensors closely involved in recognition of heat killed (HK) and live R. akari. This conclusion is supported by several lines of evidence. These include enabling cell responsiveness to HK R. akari upon complementation of TLR2/4-negative HEK293T cells with TLR2 or TLR4, compromised ability of mutants TLR2 and TLR4 to mediate NF-&#954;B activation in response to HK R. akari, and inhibition of HK R. akari-elicited TNF-&#945;secretion in human monocytes treated with blocking TLR2 or TLR4 Ab. In contrast to HK R. akari using only TLR2 and TLR4, infection with live R. akari activated IRAK1 and p38 MAPK in TLR2/4-negative HEK293T cells and showed lower TLR2/4 dependence in human monocytes and mouse macrophages, indicating the use of TLR2, TLR4, and other TLRs and/or NLRs for recognition. Experiments are ongoing to identify putative TLR2 and TLR4 agonists expressed by R. akari and to delineate innate immune sensors involved in the recognition of live bacterium.