CMV late phase-induced mTOR activation is essential for efficient virus replication in polarized human macrophages.

Human cytomegalovirus (CMV) remains one of the most important pathogens following solid-organ transplantation. Mounting evidence indicates that mammalian target of rapamycin (mTOR) inhibitors may decrease the incidence of CMV infection in solid-organ recipients. Here we aimed at elucidating the molecular mechanisms of this effect by employing a human CMV (HCMV) infection model in human macrophages, since myeloid cells are the principal in vivo targets of HCMV. We demonstrate a highly divergent host cell permissiveness for HCMV with optimal infection susceptibility in M2 but not M1 polarized macrophages. Employing an ultrahigh purified HCMV stock we observed rapamycin-independent viral entry and induction of IFN-ß transcripts, but no proinflammatory cytokines or mitogen-activated protein kinases and mTOR activation early after infection. However, in the late infection phase, sustained mTOR activation was observed in HCMV-infected cells and was required for efficient viral protein synthesis including the viral late phase proteins pUL-44 and pp65. Accordingly, rapamycin strongly suppressed CMV replication 3 and 5 days postinfection in macrophages. In conclusion, these data indicate that mTOR is essential for virus replication during late phases of the viral cycle in myeloid cells and might explain the potent anti-CMV effects of mTOR inhibitors after organ transplantation.

The multifunctional role of mTOR in innate immunity: implications for transplant immunity.

The mammalian target of rapamycin (mTOR) is an evolutionary conserved serine-threonine kinase that senses various environmental stimuli in most cells primarily to control cell growth. Restriction of cellular proliferation by mTOR inhibition led to the use of mTOR inhibitors as immunosuppressants in allogeneic transplantation as well as novel anticancer agents. However, distinct inflammatory side effects such as fever, pneumonitis, glomerulonephritis or anemia of chronic disease have been observed under this treatment regime. Apart from the mere cell-cycle regulatory effect of mTOR in dividing cells, recent data revealed a master regulatory role of mTOR in the innate immune system. Hence, inhibition of mTOR promotes proinflammatory cytokines such as IL-12 and IL-1beta, inhibits the anti-inflammatory cytokine IL-10 and boosts MHC antigen presentation via autophagy in monocytes/macrophages and dendritic cells. Moreover, mTOR regulates type I interferon production and the expression of chemokine receptors and costimulatory molecules. These results place mTOR in a complex immunoregulatory context by controlling innate and adaptive immune responses. In this review, we discuss the clinical consequences of mTOR-inhibitor therapy and aim to integrate this recent data into our current view of the molecular mechanisms of clinically employed mTOR inhibitors and discuss their relevance with special emphasis to transplantation.

Late onset Pneumocystis pneumonia in renal transplantation after long-term immunosuppression with belatacept.

Interference with T-cell function increases the risk of infections, especially during the early post-transplant period. Belatacept, a costimulation blocker, is currently being tested in phase III clinical trials. Here we report a renal transplant recipient who received belatacept and developed severe Pneumocystis jirovecii pneumonia (PCP) with fatal superinfections 4 years post transplant. Cytomegalovirus infection preceded PCP, which typically occurs in overimmunosuppressed patients, but has not yet been reported under T-cell costimulation blockade in transplant patients. This case illustrates the possibility of excessive immunosuppression even with a lymphocyte-specific regimen.

The PI3K/Akt/mTOR pathway in innate immune cells: emerging therapeutic applications.

The phosphatidylinositol-3 kinases (PI3Ks) and the mammalian target of rapamycin (mTOR) pathway have long been recognised as critically regulating metabolism, growth or survival. Recent data indicate that these molecules are also integral players in coordinating defence mechanisms in the innate immune system. In this respect, PI3K and mTOR positively regulate immune cell activation in neutrophils and mast cells. In plasmacytoid dendritic cells, these pathways have recently emerged as important regulators for type I interferon production via activation of the interferon-regulatory factor 7. Interestingly, in myeloid immune cells, PI3K and mTOR seem to constrain full immune cell activation by upregulation of the key anti-inflammatory cytokine interleukin 10 and inhibition of proinflammatory cytokines. These new insights into innate immune cell regulation may pave the way for manipulating distinct features of the innate immune system for therapeutic treatment of various inflammatory diseases and for implementation of improved vaccination strategies.

Current concepts of molecular defence mechanisms operative during urinary tract infection.

Mucosal tissues such as the gastrointestinal tract are typically exposed to a tremendous number of microorganisms and many of them are potentially dangerous to the host. In contrast, the urogenital tract is rather infrequently colonized with bacterial organisms and also devoid of physical barriers as a multi-layered mucus or ciliated epithelia, thereby necessitating separate host defence mechanisms. Recurrent urinary tract infection (UTI) represents the successful case of microbial host evasion and poses a major medical and economic health problem. During recent years considerable advances have been made in our understanding of the mechanisms underlying the immune homeostasis of the urogenital tract. Hence, the system of pathogen-recognition receptors including the Toll-like receptors (TLRs) is able to sense danger signalling and thus activate the host immune system of the genitourinary tract. Additionally, various soluble antimicrobial molecules including iron-sequestering proteins, defensins, cathelicidin and Tamm-Horsfall protein (THP), as well as their role for the prevention of UTI by modulating innate and adaptive immunity, have been more clearly defined. Furthermore, signalling mediators like cyclic adenosine monophosphate (cAMP) or the circulatory hormone vasopressin were shown to be involved in the defence of uropathogenic microbes and maintenance of mucosal integrity. Beyond this, specific receptors e.g. CD46 or beta1/beta 3-integrins, have been elucidated that are hijacked by uropathogenic E. coli to enable invasion and survival within the urogenital system paving the way for chronic forms of urinary tract infection. Collectively, the majority of these findings offer novel avenues for basic and translational research implying effective therapies against the diverse forms of acute and chronic UTI.

Toll-like receptors and chondrocytes: the lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7.

OBJECTIVE: To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)-induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes. METHODS: TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with interleukin-1beta (IL-1beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by (35)S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1beta was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis. RESULTS: TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1beta gene expression and the negative effects of LPS on matrix biosynthesis. CONCLUSION: These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.

Prevention of high-fat diet-induced adipose tissue remodeling in obese diabetic mice by n-3 polyunsaturated fatty acids.

OBJECTIVE: Obesity is associated with reduced insulin sensitivity and extensive reorganization of adipose tissue. As polyunsaturated fatty acids (PUFA) appear to inhibit diabetes development, we investigated PUFA effects on markers of matrix remodeling in white adipose tissue. METHODS AND PROCEDURE: Male obese diabetic (db/db) mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in saturated and monounsaturated fatty acids (HF/S), n-6 PUFA (HF/6) or the latter including marine n-3 PUFA (HF/3). White adipose tissue was analyzed for gene expression, fatty acid composition and by immunofluorescence. RESULTS: HF/S treatment increased adipose tissue expression of a number of genes involved in matrix degradation including matrix metalloproteinase (MMP)-12, -14 and cathepsin K, L and S compared with LF. MMP-12 gene was expressed in macrophages and adipocytes, and MMP-12 protein colocalized with both cell types. In addition, mean adipocyte area increased by 1.6-fold in HF/S-treated mice. Genes essential for collagen production, such as procollagen I, III, VI, tenascin C and biglycan were upregulated in HF/S-treated animals as well. N-3 PUFA supplementation resulted in enrichment of these fatty acids in adipose tissue. Moreover, n-3 PUFA inhibited the HF/S-induced upregulation of genes involved in matrix degradation and production I restored mean adipocyte area and prevented MMP-12 expression in macrophages and adipocytes. CONCLUSION: N-3 PUFA prevent high-fat diet-induced matrix remodeling and adipocyte enlargement in adipose tissue of obese diabetic mice. Such changes could contribute to diabetes prevention by n-3 PUFA in obese patients.

Tamm-Horsfall protein: a multilayered defence molecule against urinary tract infection.

Urinary tract infection (UTI) is the most common nonepidemic bacterial infection in humans, representing a constant danger for the host. Both innate and adaptive components of the immune system as well as stromal cells including bladder epithelium are involved in the prevention and clearance of UTI. However, the particular properties of the urogenital tract, which does not comprise typical physical barriers like a mucus or ciliated epithelium, necessitate soluble mediators with potent immunomodulatory capabilities. One candidate molecule capable of both mediating direct antimicrobial activity and alerting immune cells is the evolutionary conserved Tamm-Horsfall protein (THP). Tamm-Horsfall protein is exclusively produced by the kidney in the distal loop of Henle; however, its definite physiological function remains elusive. Mounting evidence indicates that beyond a mere direct antimicrobial activity, THP exerts potent immunoregulatory activity. Furthermore, the genetic ablation of the THP gene leads to severe infection and lethal pyelonephritis in an experimental model of UTI. Recent data are provided demonstrating that THP links the innate immune response with specific THP-directed cell-mediated immunity. In light of these novel findings we discuss the particular role of THP as a specialized defence molecule. We propose an integrated model of protective mechanisms against UTI where THP acts by two principle nonmutually exclusive mechanisms involving the capture of potentially dangerous microbes and the ability of this peculiar glycoprotein to induce robust protective immune responses against uropathogenic bacteria.