Soluble TNF‐α but not transmembrane TNF‐α sensitizes T cells for enhanced activation‐induced cell death

In addition to its proinflammatory effects, TNF‐α exhibits immunosuppression. Here, we compared the capacities of transmembrane TNF‐α (tmTNF) and soluble TNF‐α (sTNF) in regulating expansion of activated T cells by apoptosis. Splenic CD4⁺ T cells from wtTNF, TNF‐α‐deficient (TNF^?/?) and TNF^?/? mice expressing a non‐cleavable mutant tmTNF showed comparable proliferation rates upon TCR‐mediated stimulation. Activation‐induced cell death (AICD), however, was significantly attenuated in tmTNF and TNF^?/?, compared with wtTNF CD4⁺ T cells. Addition of sTNF during initial priming was sufficient to enhance susceptibility to AICD in tmTNF and TNF^?/? CD4⁺ T cells to levels seen in wtTNF CD4⁺ T cells, whereas addition of sTNF only during restimulation failed to enhance AICD. sTNF‐induced, enhanced susceptibility to AICD was dependent on both TNF receptors. The reduced susceptibility of tmTNF CD4⁺ T cells for AICD was also evident in an in vivo model of adoptively transferred CD4⁺ T‐cell‐mediated colonic inflammation. Hence, the presence of sTNF during T‐cell priming may represent an important mechanism to sensitize activated T cells for apoptosis, thereby attenuating the extent and duration of T‐cell reactivities and subsequent T‐cell‐mediated, excessive inflammation.     

The Suppressive Function of Human CD8+ iTregs is Inhibited by IL‐1β and TNFα

CD8⁺ Tregs display an immunoregulatory activity and may play an essential role in the immunopathology of several diseases. Therefore, their therapeutic potential is exquisite and further studies on their differentiation and function are essential. The aim of this study was to evaluate the role of the innate immune system in CD8⁺ iTreg differentiation and function. Naive human CD8⁺CD25^?CD45RA⁺ T cells were cultured in Treg‐inducing conditions with or without IL‐1β, TNFα or monocyte‐derived dendritic cells (DCs). The differentiation of CD8⁺CD127^?CD25^hiFoxP3^hi‐induced Tregs (CD8⁺ iTregs) is dependent on TGF‐β1 and IL‐2, which had synergistic effect upon their differentiation. CD8⁺ iTregs were also induced in a coculture with allogeneic mature DCs (mDCs). The CD8⁺ iTregs suppressive function was confirmed, which was diminished in the presence of IL‐1β and TNFα. The IL‐1β‐prevented suppressive function was associated with reduced secretion of IL‐10 and IFNγ, whereas the presence of TNFα did not affect their secretion. Furthermore, the presence of TNFα reduced IL‐10 and TGF‐β1 secretion by CD8⁺ iTregs, whereas only IL‐10 secretion was decreased by IL‐1β. Together, these results suggest that IL‐1β and TNFα prevent IL‐2‐ and TGF‐β1‐driven CD8⁺ iTregs suppressive function in human T cells. Such pro‐inflammatory innate immune response possibly mediates its negative tolerogenic effect through reduced IFNγ‐, IL‐10‐ and TGF‐β1‐driven mechanism.     

Regulation of Human Decidual Cell Macrophage Inflammatory Protein-1α (MIP-1α) Production by Inflammatory Cytokines

PROBLEM : Inflammation of human gestational tissues is a key pathophysiologic event in the genesis of infection-associated preterm labor. Human gestational tissues produce several inflammatory cytokines after stimulation with bacterial products. These include interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), and IL-6. Another class of cytokines includes chemokines of the “C-C” subclassification such as macrophage inflammatory protein-1α (MlP-1α). The purpose of this study was to determine whether cultured human decidual cells produce MIP-1α in response to other inflammatory cytokines. METHODS : Various concentrations of IL-1β, TNFα, IL-6, and IL-4 were incubated with confluent monolayer cultures of decidual cells isolated from normal term placentae for 16 h at 37°C, and MlP-1α concentrations in culture supernatants were measured by ELISA. RESULTS : We found that incubation of decidual cells with IL-1β, TNFα, and IL-4 resulted in significant concentration-dependent increases in M1P-1α production. IL-6 had no effect on MlP-1α production. CONCLUSIONS : Our data are the first to show that human decidual cells in culture produce MlP-1α in response to other inflammatory cytokines. We suggest that decidual cell production of MIP-1α is an important early event in the pathophysiology of infection-associated preterm labor.     

Soluble CD93 is an apoptotic cell opsonin recognized by αxβ2

Efferocytosis is essential for homeostasis and prevention of the inflammatory and autoimmune diseases resulting from apoptotic cell lysis. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis, with mutations in CD93 predisposing patients to efferocytosis‐associated diseases. CD93 is a cell surface protein, which is proteolytically shed under inflammatory conditions, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane‐bound form. Herein, using cell lines and human monocytes and macrophages, we demonstrate that soluble CD93 (sCD93) potently opsonizes apoptotic cells but not a broad range of microorganisms, whereas membrane‐bound CD93 has no phagocytic, efferocytic, or tethering activity. Using mass spectrometry, we identified α_xβ₂ as the receptor that recognizes sCD93, and via deletion mutagenesis determined that sCD93 binds to apoptotic cells via its C‐type lectin‐like domain and to α_xβ₂ by its EGF‐like repeats. The bridging of apoptotic cells to α_xβ₂ markedly enhanced efferocytosis by macrophages and was abrogated by α_xβ₂ knockdown. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identifies a previously unreported opsonin‐receptor system utilized by phagocytes for the efferocytic clearance of apoptotic cells.     

Anti‐retroviral activity of TRIM5α

Human immunodeficiency virus type 1 (HIV‐1) shows a very narrow host range limited to humans and chimpanzees. Experimentally, HIV‐1 does not infect Old World monkeys, such as rhesus (Rh) and cynomolgus (CM) monkeys, and fails to replicate in activated CD4 positive T lymphocytes obtained from these monkeys. In contrast, simian immunodeficiency virus isolated from a macaque monkey (SIVmac) can replicate well in both Rh and CM. In 2004, tripartite motif 5α (TRIM5α) was identified as a host factor which plays an important role in the restricted host range of HIV‐1. Rh and CM TRIM5α restrict HIV‐1 infection but not SIVmac, while in comparison, anti‐viral activity of human TRIM5α against those viruses is very weak. TRIM5α consists of the RING, B‐box 2, coiled‐coil and SPRY (B30.2) domains. The RING domain is frequently found in E3 ubiquitin ligase and TRIM5α is degraded via the ubiquitin‐proteasome pathway during HIV‐1 restriction. TRIM5α recognises the multimerised capsid (viral core) of an incoming virus by its α‐isoform specific SPRY domain and is believed to be involved in innate immunity to control retroviral infection. Differences in amino acid sequences in the SPRY domain of TRIM5α of different monkey species were found to affect species‐specific restriction of retrovirus infection, while differences in amino acid sequences in the viral capsid protein determine viral sensitivity to restriction. Accurate structural analysis of the binding surface between the viral capsid protein and TRIM5α SPRY is thus required for the development of new antiretroviral drugs that enhance anti‐HIV‐1 activity of human TRIM5α. Copyright © 2009 John Wiley & Sons, Ltd.     

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis,selectively induces pro‐inflammatory cytokines and IL‐10 and is regulated by TNF‐α, IL‐10 and IFN‐γ

Leukocyte immunoglobulin‐like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane‐bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross‐linking of LILRA5 on monocytes induced production of pro‐inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common γ chain of the FcR and LILRA5 cross‐linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro‐inflammatory cytokines as well as IL‐10. LILRA5 mRNA and protein expression was tightly regulated by TNF‐α, IL‐10 and IFN‐γ. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.     

Type I NKT‐cell‐mediated TNF‐α is a positive regulator of NLRP3 inflammasome priming

The NLRP3 inflammasome plays a crucial role in the innate immune response to pathogens and exogenous or endogenous danger signals. Its activity must be precisely and tightly regulated to generate tailored immune responses. However, the immune cell subsets and cytokines controlling NLRP3 inflammasome activity are still poorly understood. Here, we have shown a link between NKT‐cell‐mediated TNF‐α and NLRP3 inflammasome activity. The NLRP3 inflammasome in APCs was critical to potentiate NKT‐cell‐mediated immune responses, since C57BL/6 NLRP3 inflammasome‐deficient mice exhibited reduced responsiveness to α‐galactosylceramide. Importantly, NKT cells were found to act as regulators of NLRP3 inflammasome signaling, as NKT‐cell‐derived TNF‐α was required for optimal IL‐1β and IL‐18 production by myeloid cells in response to α‐galactosylceramide, by acting on the NLRP3 inflammasome priming step. Thus, NKT cells play a role in the positive regulation of NLRP3 inflammasome priming by mediating the production of TNF‐α, thus demonstrating another means by which NKT cells control early inflammation.     

Antibodies Against Sporothrix schenckii Enhance TNF‐α Production and Killing by Macrophages

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell‐mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti‐gp70. Additionally, we show an increase in the levels of pro‐inflammatory cytokines such as TNF‐α and IL‐1β. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF‐α production and fungus killing by macrophages in experimental sporotrichosis.     

Keratinocyte Growth Factor Stimulates Macrophage Inflammatory Protein 3α and Keratinocyte‐derived Chemokine Secretion by Mouse Uterine Epithelial Cells

Citation Haddad SN, Wira CR. Keratinocyte growth factor stimulates macrophage inflammatory protein 3α and keratinocyte‐derived chemokine secretion by mouse uterine epithelial cells. Am J Reprod Immunol 2010; 64: 197–211 Problem Communication between uterine epithelial cells and the underlying stromal fibroblasts is critical for proper endometrial function. Stromal fibroblast‐derived growth factors have been shown to regulate epithelial immune functions. The purpose of this study was to determine whether keratinocyte growth factor (KGF) regulates uterine epithelial cell chemokine and antimicrobial secretion. Method of study Uterine epithelial cells were isolated from Balb/c mice and cultured in either 96‐well plates or transwell inserts. Epithelial cells were treated with KGF, epidermal growth factor (EGF), or hepatocyte growth factor (HGF). Macrophage inflammatory protein 3α (MIP3α) and keratinocyte‐derived chemokine (KC) levels were measured by ELISA. Results Keratinocyte growth factor stimulated the secretion of MIP3α and KC. The effects on MIP3α by KGF were specific because EGF and HGF had no effect. In contrast, KGF, EGF, and HGF had similar effects on KC. Furthermore, KGF administered to the apical side of epithelial cells had no effect on MIP3α or KC secretion, indicating that the KGF receptor is located on the basolateral surface of uterine epithelial cells. Conclusion We demonstrate that KGF plays a role in uterine epithelial cell secretion of MIP3α and KC, key immune mediators involved in the protection of mucosal surfaces in the female reproductive tract.     

Corticosteroid enhances TNF‐α‐mediated leukocyte adhesion to pulmonary microvascular endothelial cells

Background: Some severe asthma patients are characterized by elevated levels of tumor necrosis factor alpha (TNF‐α) and neutrophilic inflammation in the airways. Although such phenotypic changes in asthma might contribute to corticosteroid refractoriness, the role of TNF‐α in the process remains unclear. TNF‐α exerts its biological effects mainly by acting on the vascular endothelium, and thereby upregulates leukocyte recruitment into inflamed tissues. The aim of this study was to investigate the effects of dexamethasone (DEX) on the TNF‐α‐mediated responses of human microvascular endothelial cells from lung blood vessels (HMVEC‐LBl) in vitro. Methods: HMVEC‐LBl were cultured with TNF‐α in the presence and absence of DEX. The effects of DEX on various TNF‐α‐mediated responses, such as the expressions of chemokines and cellular adhesion molecules, leukocyte adhesion were determined. Results: TNF‐α significantly induced growth‐related oncogene alpha (GRO‐α), interleukin 8 (IL‐8), regulated on activation, normal T‐cell expressed and secreted (RANTES) and interferon‐inducible protein 10 (IP‐10) productions and cell surface expressions of intracellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) on HMVEC‐LBl. TNF‐α‐induced GRO‐α and IL‐8 were slightly attenuated by DEX treatment (reaches to 89% and 79%, respectively), whereas expressions of IP‐10, ICAM‐1 and VCAM‐1 were significantly enhanced by the same treatment (up to 172%, 152% and 139%, respectively). Correspondingly, in vitro adhesion of eosinophils and neutrophils to TNF‐α‐treated HMVEC‐LBl were significantly enhanced by DEX. Conclusions: Some proinflammatory effects of DEX, a corticosteroid, were found in TNF‐α‐mediated in vitro reactions of pulmonary microvascular endothelial cells, i.e. chemokine productions and leukocyte adhesion. These in vitro results may explain, at least in part, the corticosteroid refractoriness accompanied by a marked increase in TNF‐α production that is seen in severe asthmatic patients.     

The Synergy of ‐260T T CD14 and ‐308GG TNF‐α Genotypes in Survival of Critically Ill Patients

Literature suggests that the analysis of several polymorphic genetic markers is more informative than the analysis of a single polymorphism. In this study, we tested whether the shared inheritance of TLR2 and TLR4 and TNF‐α allelic variants may act in synergy with ‐260C>T CD14 SNP on the outcome from critical conditions. We monitored 524 critically ill patients from South Brazilian, daily from the ICU admission to their discharge from hospital, or death. Our results revealed that TLR2, TLR4 or TNF‐α SNPs alone did not show a significant role in the outcome from critical illness. However, when we performed a combined analysis with the CD14 inheritance, we detected a significant higher survivor rate in ‐260TT CD14/‐308GG TNF‐α individuals (P = 0.037). In the adjusted analysis including the main clinical predictors to mortality, we observed that ‐260TT/‐308GG double‐genotype was a significant protective factor towards survival (P = 0.046). An increased probability for survival of ‐260TT/‐308GG was also observed by ‘pathway genetic load’ analysis (unweighted: P = 0.041; weighted: P = 0.036). When we applied a hazard function analysis with the ‐260TT/‐308GG variable as a discriminating factor, ‐260TT/‐308GG patients group had, in fact, a higher survivor rate (P = 0.024). Connected to the beneficial effect of ‐260TT CD14, the ‐308GG TNF‐α genotype was protective against the reported over expression of TNF‐α caused by ‐308A rare allele. Results support the hypothesis that the interaction between ‐260C>T CD14 and ‐308G>A TNF‐α functional SNPs may be synergistically influencing the outcome of critically ill patients.     

The Peritoneal Leptin,MCP‐1 and TNF‐α in the Pathogenesis of Endometriosis‐Associated Infertility

Citation Tao Y, Zhang Q, Huang W, Zhu H, Zhang D, Luo W. The peritoneal leptin, MCP‐1 and TNF‐α in the pathogenesis of endometriosis‐associated infertility. Am J Reprod Immunol 2011; 65: 403–406 Problem To explore the roles of leptin, monocyte chemotactic protein (MCP)‐1, and tumour necrosis factor (TNF)‐α in the peritoneal fluid (PF) in the pathogenesis of endometriosis‐associated infertility. Method of study Leptin, MCP‐1, and TNF‐α levels in the PF from 28 infertile women with endometriosis (study group), 23 women with fallopian‐associated infertility (controls), and 24 women with myoma (controls) were determined by performing enzyme‐linked immunosorbent assay (ELISA). Result Leptin and TNF‐α levels in the PF showed no significant difference among three groups. The MCP‐1 level in patients with endometriosis was higher than those in fallopian‐associated infertility group and myoma group (P < 0.01). There was a positive correlation between leptin and MCP‐1 levels in the PF of patients with endometriosis (P < 0.05). Conclusion Peritoneal leptin and MCP‐1 play important roles in the pathogenesis of infertility in the early stage of endometriosis.     

Genetic polymorphisms of tumour necrosis factor alpha (TNF‐α) promoter gene and response to TNF‐α inhibitors in Spanish patients with inflammatory bowel disease

Tumour necrosis factor alpha (TNF‐α) has an important role in inflammatory response. Alterations in the regulation of TNF‐α have been implicated in a variety of inflammatory disorders, including Inflammatory bowel disease (IBD). Indeed, a common treatment for IBD is the use of TNF‐α inhibitors. Polymorphisms in the TNF‐α promoter region are known to affect the level of gene expression. Our aim was to investigate the influence of these single nucleotide polymorphisms (SNPs) in TNF‐α promoter gene play in the risk of IBD in a Spanish population and their individual response to anti‐TNF‐α treatment. DNA samples from patients with IBD and controls were screened for TNF‐α ?238G/A (rs361525) and ?308G/A (rs1800629) SNPs by PCR‐SSOP using a microbeads luminex assay and compared with response to TNF‐α inhibitors. There were not statistical differences in ?238G/A and ?308G/A allele and genotype frequencies between patients. However, we found an increased frequency of ?308A allele and ?308GA genotype in these nonresponders patients to TNF‐α inhibitors with respect to responders patients (Pc < 0.05). This ?308GA genotype has been classified as high producer of this cytokine. This fact could actually be interesting to explain the different response of patients with IBD with respect to TNF‐α inhibitors. TNF‐α promoter gene polymorphism does not seem to play a role in IBD susceptibility, but particular TNF‐α genotypes may be involved in the different responses to TNF‐α inhibitor treatment in Spanish patients with IBD.     

Gx‐50 reduces β‐amyloid‐induced TNF‐α, IL‐1β, NO,and PGE2 expression and inhibits NF‐κB signaling in a mouse model of Alzheimer's disease

Chronic inflammation, which is regulated by overactivated microglia in the brain, accelerates the occurrence and development of Alzheimer's disease (AD). Gx‐50 has been investigated as a novel drug for the treatment of AD in our previous studies. Here, we investigated whether gx‐50 possesses anti‐inflammatory effects in primary rat microglia and a mouse model of AD, amyloid precursor protein (APP) Tg mice. The expression of TNF‐α, IL‐1β, NO, prostaglandin E2, and the expression of iNOS and COX2 were inhibited by gx‐50 in amyloid β (Aβ) treated rat microglia; additionally, microglial activation and the expression of IL‐1β, iNOS, and COX2 were also significantly suppressed by gx‐50 in APP⁺ transgenic mice. Furthermore, gx‐50 inhibited the activation of NF‐κB and MAPK cascades in vitro and in vivo in APP‐Tg mice. Moreover, the expression of TLR4 and its downstream signaling proteins MyD88 and tumor necrosis factor receptor associated factor 6 (TRAF6) was reduced by gx‐50 in vitro and in vivo. Interestingly, silencing of TLR4 reduced Aβ‐induced upregulation of IL‐1β and TRAF6 to levels similar to gx‐50 inhibition; moreover, overexpression of TLR4 increased the expression of MyD88 and TRAF6, which was significantly reduced by gx‐50. These findings provide strong evidence that gx‐50 has anti‐inflammatory effects against Aβ‐triggered microglial overactivation via a mechanism that involves the TLR4‐mediated NF‐κBB/MAPK signaling cascade.