Cutting edge: impaired Toll-like receptor expression and function in aging

Toll-like receptors (TLR) are pattern recognition receptors that recognize conserved molecular patterns on microbes and link innate and adaptive immune systems. We investigated whether the enhanced susceptibility to bacterial, yeast, and viral infections and poor adaptive immune responses in aging are a result of diminished expression and function of TLRs. We examined the expression and function of all murine TLRs on macrophages from young and aged mice. Both splenic and activated peritoneal macrophages from aged mice expressed significantly lower levels of all TLRs. Furthermore, macrophages from aged mice secreted significantly lower levels of IL-6 and TNF-alpha when stimulated with known ligands for TLR1 and 2, 2 and 6,TLR3, TLR4, TLR5, and TLR9 when compared with those from young mice. These results support the concept that increased susceptibility to infections and poor adaptive immune responses in aging may be due to the decline in TLR expression and function.     

Cutting edge: Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent

The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-kappaB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.     

Cutting edge: Th2 cell trafficking into the allergic lung is dependent on chemoattractant receptor signaling

Th2 cells are recruited to the lung where they mediate the asthma phenotype. Since the molecular mechanisms regulating Th2 cell trafficking remain unknown, we sought to determine whether trafficking of Th2 cells into the lung is mediated by G alpha i-coupled chemoattractant receptors. We show here that in contrast to untreated Th2 cells, pertussis toxin-treated Th2 cells were unable to traffic into the lung, airways, or lymph nodes following Ag challenge and therefore were unable to induce allergic inflammation in vivo. Pertussis toxin-treated Th2 cells were functional cells, however, and when directly instilled into the airways of mice, bypassing their need to traffic to the lung, were able to induce airway eosinophilic inflammation. These studies conclusively demonstrate that trafficking of Th2 cells into the lung is an active process dependent on chemoattractant receptors.     

Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins

The Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns (PAMPs). TLR2 is essential for the signaling of a variety of PAMPs, including bacterial lipoprotein/lipopeptides, peptidoglycan, and GPI anchors. TLR6 associates with TLR2 and recognizes diacylated mycoplasmal lipopeptide along with TLR2. We report here that TLR1 associates with TLR2 and recognizes the native mycobacterial 19-kDa lipoprotein along with TLR2. Macrophages from TLR1-deficient (TLR1(-/-)) mice showed impaired proinflammatory cytokine production in response to the 19-kDa lipoprotein and a synthetic triacylated lipopeptide. In contrast, TLR1(-/-) cells responded normally to diacylated lipopeptide. TLR1 interacts with TLR2 and coexpression of TLR1 and TLR2 enhanced the NF-kappaB activation in response to a synthetic lipopeptide. Furthermore, lipoprotein analogs whose acylation was modified were preferentially recognized by TLR1. Taken together, TLR1 interacts with TLR2 to recognize the lipid configuration of the native mycobacterial lipoprotein as well as several triacylated lipopeptides.     

Cutting edge: a Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling

Toll-like receptors (TLRs) are key mediators of the innate immune response to microbial pathogens. We investigated the role of TLRs in the recognition of Mycobacterium leprae and the significance of TLR2Arg(677)Trp, a recently discovered human polymorphism that is associated with lepromatous leprosy. In mice, TNF-alpha production in response to M. leprae was essentially absent in TLR2-deficient macrophages. Similarly, human TLR2 mediated M. leprae-dependent activation of NF-kappaB in transfected Chinese hamster ovary and human embryonic kidney 293 cells, with enhancement of this signaling in the presence of CD14. In contrast, activation of NF-kappaB by human TLR2Arg(677)Trp was abolished in response to M. leprae and Mycobacterium tuberculosis. The impaired function of this TLR2 variant provides a molecular mechanism for the poor cellular immune response associated with lepromatous leprosy and may have important implications for understanding the pathogenesis of other mycobacterial infections.     

Cutting edge: differential expression of chemokines in Th1 and Th2 cells is dependent on Stat6 but not Stat4

The in vivo function of Th cell subsets is largely dependent on the ability of differentiated CD4+ T cells to be recruited to specific sites and secrete restricted sets of cytokines. In this paper we demonstrate that Th1 and Th2 cells secrete discrete patterns of chemokines, small m.w. cytokines that function as chemoattractants in inflammatory reactions. Th2 cells secrete macrophage-derived chemokine and T cell activation gene 3, and acquisition of this pattern of expression is dependent on Stat6. In contrast, Th1 cells secrete lymphotactin and RANTES, though unlike IFN-gamma, expression of these chemokines is independent of Stat4. We further show that supernatants from activated Th2 cells preferentially induce the chemotaxis of Th2 over Th1 cells, corresponding with Stat6-dependent expression of CCR4 and CCR8 in Th2 cells. These data provide the basis for restricted and direct T cell-mediated cellular recruitment to sites of inflammation.     

Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin

Host defense against intracellular pathogens depends upon innate and adaptive antimicrobial effector pathways. TLR2/1-activation of monocytes leads to the vitamin D-dependent production of cathelicidin and, at the same time, an antimicrobial activity against intracellular Mycobacterium tuberculosis. To determine whether induction of cathelicidin was required for the vitamin D-triggered antimicrobial activity, the human monocytic cell line THP-1 was infected with M. tuberculosis H37Ra and then activated with the active vitamin D hormone 1,25-dihydroxyvitamin D(3) (1,25D(3)). 1,25D(3) stimulation resulted in antimicrobial activity against intracellular M. tuberculosis and expression of cathelicidin mRNA and protein. Using small interfering RNA (siRNA) specific for cathelicidin, 1,25D(3)-induced cathelicidin mRNA and protein expressions were efficiently knocked down, whereas a nonspecific siRNA control had little effect. Finally, 1,25D(3)-induced antimicrobial activity was completely inhibited in the presence of siRNA against cathelicidin, instead leading to enhanced intracellular growth of mycobacteria. These data demonstrate that cathelicidin is required for the 1,25D(3)-triggered antimicrobial activity against intracellular M. tuberculosis.     

Cutting edge: tumor rejection mediated by NKG2D receptor-ligand interaction is dependent upon perforin

We have investigated the primary immunity generated in vivo by MHC class I-deficient and -competent tumor cell lines that expressed the NKG2D ligand retinoic acid early inducible-1 (Rae-1) beta. Rae-1beta expression on class I-deficient RMA-S lymphoma cells enhanced primary NK cell-mediated tumor rejection in vivo, whereas RMA-Rae-1beta tumor cells were rejected by a combination of NK cells and CD8(+) T cells. Rae-1beta expression stimulated NK cell cytotoxicity and IFN-gamma secretion in vitro, but not proliferation. Surprisingly, only NK cell perforin-mediated cytotoxicity, but not production of IFN-gamma, was critical for the rejection of Rae-1beta-expressing tumor cells in vivo. This distinct requirement for perforin activity contrasts with the NK cell-mediated rejection of MHC class I-deficient RMA-S tumor cells expressing other activating ligands such as CD70 and CD80. Thus, these results indicated that NKG2D acted as a natural cytotoxicity receptor to stimulate perforin-mediated elimination of ligand-expressing tumor cells.     

Cutting edge: nitrogen bisphosphonate-induced inflammation is dependent upon mast cells and IL-1

Nitrogen-containing bisphosphonates (NBPs) are taken by millions for bone disorders but may cause serious inflammatory reactions. In this study, we used a murine peritonitis model to characterize the inflammatory mechanisms of these agents. At dosages comparable to those used in humans, injection of NBPs into the peritoneum caused recruitment of neutrophils, followed by an influx of monocytes. These cellular changes corresponded to an initial increase in IL-1α, which preceded a rise in multiple other proinflammatory cytokines. IL-1R, IL-1α, and IL-1β were required for neutrophil recruitment, whereas other MyD88-dependent signaling pathways were needed for the monocyte influx. Mice deficient in mast cells, but not mice lacking lymphocytes, were resistant to NBP-induced inflammation, and reconstitution of these mice with mast cells restored sensitivity to NBPs. These results document the critical role of mast cells and IL-1 in NBP-mediated inflammatory reactions.     

Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma

Recognition of microbial components by APCs and their activation through Toll-like receptors (TLR) leads to the induction of adaptive immune responses. In this study, we show that activation of TLR2 by its synthetic ligand Pam3Cys, in contrast to activation of TLR9 by immunostimulatory DNA (ISS-ODN), induces a prominent Th2-biased immune response. Activation of APCs by Pam3Cys resulted in the induction of Th2-associated effector molecules like IL-13, and IL-1beta, GM-CSF and up-regulation of B7RP-1, but low levels of Th1-associated cytokines (IL-12, IFNalpha, IL-18, IL-27). Accordingly, TLR2 ligands aggravated experimental asthma. These data indicate that the type of TLR stimulation during the initial phase of immune activation determines the polarization of the adaptive immune response and may play a role in the initiation of Th2-mediated immune disorders, such as asthma.     

Cutting edge: Inhibition of NF-kappaB-mediated TSLP expression by retinoid X receptor

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) has important roles in the initiation of allergic airway inflammation and the activation of dendritic cells. We have shown that the human TSLP gene is regulated in a NF-kappaB-dependent manner; however the factors that negatively regulate TSLP expression are not known. In this study we demonstrate that 9-cis-retinoic acid (9-cis-RA) is a negative regulator of TSLP expression in airway epithelial cells. This inhibition is manifested as a block in the IL-1beta-mediated recruitment of NF-kappaB to the human TSLP promoter. 9-cis-RA-mediated inhibition is not restricted to TSLP gene expression but rather reflects a general inhibition of NF-kappaB activation, as other NF-kappaB-regulated-genes were also inhibited in a similar manner by 9-cis-RA treatment. Taken as a whole, these data demonstrate that inhibition of IL-1beta-dependent genes by active retinoid X receptors involves antagonism of NF-kappaB signaling.     

Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2.

The agent of Lyme disease, Borrelia burgdorferi, produces membrane lipoproteins possessing potent inflammatory properties linked to disease pathology. The recent association of toll-like receptors (TLR) 2 and 4 with LPS responses prompted the examination of TLR involvement in lipoprotein signaling. The ability of human cell lines to respond to lipoproteins was correlated with the expression of TLR2. Transfection of TLR2 into cell lines conferred responsiveness to lipoproteins, lipopeptides, and sonicated B. burgdorferi, as measured by nuclear translocation of NF-kappaB and cytokine production. The physiological importance of this interaction was demonstrated by the 10-fold greater sensitivity of TLR2-transfected cells to lipoproteins than LPS. Futhermore, TLR2-dependent signaling by lipoproteins was facilitated by CD14. These data indicate that TLR2 facilitates the inflammatory events associated with Lyme arthritis. In addition, the widespread expression of lipoproteins by other bacterial species suggests that this interaction may have broad implications in microbial inflammation and pathogenesis.     

Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.     

Cutting edge: differentiation of antitumor CTL in vivo requires host expression of Stat1.

Several lines of evidence suggest that an IFN-gamma-producing, Th1/Tc1 phenotype may be optimal for tumor rejection. Recent work has indicated that IFN signaling on tumor cells is important for protection against carcinogenesis. However, the potential involvement of IFN signaling among host immune cells has not been carefully examined. To this end, Stat1-deficient mice were employed as tumor recipients. In contrast to wild-type mice, Stat1-/- mice failed to reject immunogenic tumors and did not support regression of poorly immunogenic tumors when treated with an IL-12-based vaccine. T cells from immunized Stat1-/- mice produced 50% of the levels of IFN-gamma and lacked cytolytic activity compared with wild-type mice, and NK lytic activity also was not observed. Lack of cytolytic function correlated with a failure to up-regulate serine esterase activity. Thus, IFN-mediated signaling on host cells is required for the development of antitumor lytic effector cells.