IntroductionMesenchymal stem cells (MSCs) comprise an adult population that resides in many organs and exhibits multiple functions and phenotypes upon in vitro culture; MSCs can be induced to differentiate into mesodermal cell lineages, 1,2 support and regulate hematopoiesis, [3][4][5][6][7] regulate the stem-cell niche, [8][9][10][11][12] and may participate in the repair of tissue damage inflicted by normal wear and tear, injury, or disease. [13][14][15][16] MSCs comprise 0.01% to 0.001% of the bone marrow (BM)-nucleated cells and are obtained by expansion of the BM, plastic-adherent cell fraction. 1,[17][18][19][20][21] Under certain physiologic or experimental conditions, MSCs can be induced to differentiate in vitro into cells of the mesodermal lineage, specifically to osteocytes, adipocytes, chondrocytes, myocytes, tenocytes, myocardiocytes, and hematopoietic supportive stroma. 1,17,19,22 MSCs are an attractive cell-based therapy tool for developmental defects; degenerating diseases; and bone, cartilage, muscle, and other mesodermal tissue injuries. [23][24][25][26][27][28][29][30] Toll-like receptors (TLRs) are a class of molecules first discovered to play a role in body development 31 and later found to play a role in body maintenance. [32][33][34][35][36] The TLR family has been shown to be of importance in the innate immune system for the recognition of pathogen-associated molecular patterns (PAMPs) by immune cells, initiating a primary response toward invading pathogens and recruitment of the adaptive immune response. 32,[37][38][39][40][41][42][43][44][45][46][47][48][49] TLRs can be activated not only by pathogen components, but also by mammalian endogenous molecules such as heat-shock proteins and extracellular matrix breakdown products. [50][51][52] In the steady state, during the generation of immune cells, as well as under pathologic conditions, there are intimate interactions between lymphocyte populations and the organ stroma mesenchyme. These interactions regulate cell growth and differentiation and control cell functions. It is possible therefore that lymphocytes and the stromal mesenchyme share regulatory mechanisms. To test this possibility we aimed, in the present study, to examine the expression and possible regulatory functions of TLRs in mesenchymal cells.We explored the expression of TLRs by MSCs, the response of MSCs to known TLR activators, and the ability of a TLR-2 ligand to regulate MSC proliferation and differentiation. We show here that cultured MSCs express TLR molecules 1 to 8, but not TLR-9. Activation of MSCs by TLR ligands induced interleukin-6 (IL-6) secretion and nuclear factor B (NF-B) nuclear translocation. Pam3Cys, a prototypic ligand for TLR-2, induced proliferation of MSCs and regulated their differentiation. Relatively little is known about the signals that regulate MSC proliferation, differentiation, and development. 53,54 Our findings suggest that TLR signaling may play a role in restraining MSC differentiation and thus promote MSC renewal. Materials and methods ...

We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-γ by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses.

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1α (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.

We recently reported that heat shock protein 60 (HSP60) via TLR4 signaling activates B cells and induces them to proliferate and secrete IL-10. We now report that HSP60 inhibits mouse B cell apoptosis, spontaneous or induced by dexamethasone or anti-IgM activation. Unlike HSP60 enhancement of B cell proliferation and IL-10 secretion, TLR4 signaling was not required for the inhibition of apoptosis by HSP60; nevertheless, MyD88 was essential. Inhibition of apoptosis by HSP60 was associated with up-regulation of the antiapoptotic molecules Bcl-2, Bcl-xL, and survivin, maintenance of the mitochondrial transmembrane potential, and inhibition of caspase-3 activation. Moreover, B cells incubated with HSP60 manifested prolonged survival following transfer into recipient mice. These results extend the varied role of HSP60 in the innate regulation of the adaptive immune response.

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.