Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.     

Neutrophils as firemen, production of anti-inflammatory mediators by neutrophils in a mixed cell environment

Neutrophils represent critical components of the innate immune system that bear primary responsibility for phagocytosis and killing of invading pathogens. Following stimulation of human whole blood, robust production of multiple cytokines and cytokine inhibitors occurs. We attempted to define the cell population responsible for the synthesis of different mediators by first stimulating whole blood and then isolating pure populations of granulocytes and monocytes. Monocytes produced mRNA coding for the classic pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6, while mRNA for these cytokines was not detectable in the isolated neutrophils. In contrast, neutrophils produce significant quantities of cytokine inhibitors such as the type 2 TNF soluble receptor and the IL-1 receptor antagonist. Both neutrophils and monocytes produced mRNA coding for IL-8. These data indicate that following stimulation of a mixed cell population the monocytes primarily produce pro-inflammatory mediators while the neutrophils synthesize a significant portion of the anti-inflammatory mediators. The neutrophils may be compared to firemen who bring the resources necessary to put out the flame of acute inflammation.     

CrmE, a novel soluble tumor necrosis factor receptor encoded by poxviruses

Cytokines and chemokines play a critical role in both the innate and acquired immune responses and constitute prime targets for pathogen sabotage. Molecular mimicry of cytokines and cytokine receptors is a mechanism encoded by large DNA viruses to modulate the host immune response. Three tumor necrosis factor receptors (TNFRs) have been identified in the poxvirus cowpox virus. Here we report the identification and characterization of a fourth distinct soluble TNFR, named cytokine response modifier E (CrmE), encoded by cowpox virus. The crmE gene has been sequenced in strains of the orthopoxviruses cowpox virus, ectromelia virus, and camelpox virus, and was found to be active in cowpox virus. crmE is expressed as a secreted 18-kDa protein with TNF binding activity. CrmE was produced in the baculovirus and vaccinia virus expression systems and was shown to bind human, mouse, and rat TNF, but not human lymphotoxin alpha, conjugates of lymphotoxins alpha and beta, or seven other ligands of the TNF superfamily. However, CrmE protects cells only from the cytolytic activity of human TNF. CrmE is a new member of the TNFR superfamily which is expressed as a soluble molecule that blocks the binding of TNF to high-affinity TNFRs on the cell surface. The remarkable finding of a fourth poxvirus-encoded TNFR suggests that modulation of TNF activity is complex and represents a novel viral immune evasion mechanism.     

Potent Anti-Inflammatory Activity of Novel Microtubule-Modulating Brominated Noscapine Analogs

Noscapine, a plant-derived, non-toxic, over-the-counter antitussive alkaloid has tubulin-binding properties. Based upon the structural resemblance of noscapine to colchicine, a tubulin-binding anti-inflammatory drug, noscapine and its semi-synthetic brominated analogs were examined for in vitro anti-inflammatory activity. Brominated noscapine analogs were found to inhibit cytokine and chemokine release from macrophage cell lines but did not affect cell viability. Brominated noscapine analogs demonstrated anti-inflammatory properties in both TLR- and non-TLR induced in vitro innate immune pathway inflammation models, mimicking septic and sterile infection respectively. In addition, electron microscopy and immunoblotting data indicated that these analogs induced robust autophagy in human macrophages. This study is the first report to identify brominated noscapines as innate immune pathway anti-inflammatory molecules.     

Novel mutants of human tumor necrosis factor with dominant-negative properties

Tumor necrosis factor (TNF) is a polyfunctional cytokine, one of the key mediators of inflammation and innate immunity. On the other hand, systemic or local TNF overexpression is typical of such pathological states as rheumatoid arthritis, psoriasis, Crohn’s disease, septic shock, and multiple sclerosis. Neutralization of TNF activity has a marked curative effect for some diseases; therefore, the search for various TNF blockers is a promising field of protein engineering and biotechnology. According to the previously developed concept concerning the possibility of designing dominant-negative mutants, the following TNF variants have been studied: TNFY87H + A145R, TNFY87H + A96S + A145R, and TNFV91N + A145R. All of these form inactive TNF heterotrimers with the native protein. The ability of mutants to neutralize the effect of TNF was investigated. The addition of mutants to the native protein was shown to provide a concentration-dependent suppression of TNF cytotoxicity against the mouse fibroblast cell line L929. Thus, novel inhibitors of human TNF can be engineered on the basis of these muteins.     

Pentraxins in innate immunity: lessons from PTX3

The innate immune system constitutes the first line of defence against microorganisms and plays a primordial role in the activation and regulation of adaptive immunity. The innate immune system is composed of a cellular arm and a humoral arm. Components of the humoral arm include members of the complement cascade and soluble pattern recognition molecules (PRMs). These fluid-phase PRMs represent the functional ancestors of antibodies and play a crucial role in the discrimination between self, non-self and modified-self. Moreover, evidence has been presented that these soluble PRMs participate in the regulation of inflammatory responses and interact with the cellular arm of the innate immune system. Pentraxins consist of a set of multimeric soluble proteins and represent the prototypic components of humoral innate immunity. Based on the primary structure of the protomer, pentraxins are divided into two groups: short pentraxins and long pentraxins. The short pentraxins C-reactive protein and serum amyloid P-component are produced by the liver and represent the main acute phase proteins in human and mouse, respectively. The long pentraxin PTX3 is produced by innate immunity cells (e.g. PMN, macrophages, dendritic cells), interacts with several ligands and plays an essential role in innate immunity, tuning inflammation and matrix deposition. PTX3 provides a paradigm for the mode of action of humoral innate immunity.     

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis

Background

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.

Methods

The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.

Results

We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.

Conclusions

The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.

General significance

Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.     

Role of interleukin-13 in innate and adaptive immunity.

Initially thought to be functionally redundant with IL-4 as a predominant anti-inflammatory factor secreted during type-2 T-cell responses, IL-13 possesses a number of additional properties that distinguish it from IL-4 in addition to having both anti-inflammatory and immune activating properties. This review centers primarily on the role of IL-13 in the regulation of cellular functions of innate immunity and acquired immunity against certain microbial pathogens. First, we discuss IL-13's regulation of innate cell targets and its impact on inflammation, antigen uptake and antigen presentation. Second, we focus on IL-13's involvement in acquired immunity to infectious helminths and protozoa. The role of this cytokine in immune responses is still being determined but evidence to date suggests this molecule has been conserved as an important regulatory factor involved in both early innate and late adaptive responses.     

SOCS2-induced proteasome-dependent TRAF6 degradation: a common anti-inflammatory pathway for control of innate immune responses

Pattern recognition receptors and receptors for pro-inflammatory cytokines provide critical signals to drive the development of protective immunity to infection. Therefore, counter-regulatory pathways are required to ensure that overwhelming inflammation harm host tissues. Previously, we showed that lipoxins modulate immune response during infection, restraining inflammation during infectious diseases in an Aryl hydrocarbon receptor (AhR)/suppressors of cytokine signaling (SOCS)2-dependent-manner. Recently, Indoleamine-pyrrole 2,3- dioxygenase (IDO)-derived tryptophan metabolites, including L-kynurenine, were also shown to be involved in several counter-regulatory mechanisms. Herein, we addressed whether the intracellular molecular events induced by lipoxins mediating control of innate immune signaling are part of a common regulatory pathway also shared by L-kynurenine exposure. We demonstrate that Tumor necrosis factor receptor-associated factor (TRAF)6--member of a family of adapter molecules that couple the TNF receptor and interleukin-1 receptor/Toll-like receptor families to intracellular signaling events essential for the development of immune responses--is targeted by both lipoxins and L-kynurenine via an AhR/SOCS2-dependent pathway. Furthermore, we show that LXA?- and L-kynurenine-induced AhR activation, its subsequent nuclear translocation, leading SOCS2 expression and TRAF6 Lys47-linked poly-ubiquitination and proteosome-mediated degradation of the adapter proteins. The in vitro consequences of such molecular interactions included inhibition of TLR- and cytokine receptor-driven signal transduction and cytokine production. Subsequently, in vivo proteosome inhibition led to unresponsiveness to lipoxins, as well as to uncontrolled pro-inflammatory reactions and elevated mortality during toxoplasmosis. In summary, our results establish proteasome degradation of TRAF6 as a key molecular target for the anti-inflammatory pathway triggered by lipoxins and L-kynurenine, critical counter-regulatory mediators in the innate and adaptive immune systems.     

Innate and adaptive immune responses in asthma

The recognition that asthma is primarily an inflammatory disorder of the airways associated with T helper type 2 (T(H)2) cell-dependent promotion of IgE production and recruitment of mast cells and eosinophils has provided the rationale for disease control using inhaled corticosteroids and other anti-inflammatory drugs. As more has been discovered about the cytokine, chemokine and inflammatory pathways that are associated with T(H)2-driven adaptive immunity, attempts have been made to selectively inhibit these in the hope of discovering new therapeutics as predicted from animal models of allergic inflammation. The limited success of this approach, together with the recognition that asthma is more than allergic inflammation, has drawn attention to the innate immune response in this disease. Recent advances in our understanding of the sentinel role played by innate immunity provides new targets for disease prevention and treatment. These include pathways of innate stimulation by environmental or endogenous pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) to influence the activation and trafficking of DCs, innate sources of cytokines, and the identification of new T cell subsets and lymphoid cells.     

Modulation of tumor necrosis factor and interleukin-1-dependent NF-kappaB activity by mPLK/IRAK

The innate immune response is an important defense against pathogenic agents. A component of this response is the NF-kappaB-dependent activation of genes encoding inflammatory cytokines such as interleukin-8 (IL-8) and cell adhesion molecules like E-selectin. Members of the serine/threonine innate immune kinase family of proteins have been proposed to mediate the innate immune response. One serine/threonine innate immune kinase family member, the mouse Pelle-like kinase/human interleukin-1 receptor-associated kinase (mPLK/IRAK), has been proposed to play an obligate role in promoting IL-1-mediated inflammation. However, it is currently unknown whether mPLK/IRAK catalytic activity is required for IL-1-dependent NF-kappaB activation. The present study demonstrates that mPLK/IRAK catalytic activity is not required for IL-1-mediated activation of an NF-kappaB-dependent signal. Intriguingly, catalytically inactive mPLK/IRAK inhibits type 1 tumor necrosis factor (TNF) receptor-dependent NF-kappaB activation. The pathway through which mPLK/IRAK mediates this TNF response is TRADD- and TRAF2-independent. Our data suggest that in addition to its role in IL-1 signaling, mPLK/IRAK is a component of a novel signal transduction pathway through which TNF R1 activates NF-kappaB-dependent gene expression.     

Tumour necrosis factor and cancer

TNF is a cytokine whose diverse actions are dependent on the local microenvironment. As a member of the cytokine network, TNF plays an important role in infection and inflammation, but excessive and deregulated production can contribute to disease processes. Likewise in malignant disease, TNF may have a role in cancer therapy and contribute to host response against tumours, but it may also be involved in the progression and spread of the cancer. In experimental models, recombinant TNF can induce significant haemorrhagic necrosis, localised to the tumour vasculature and specific tumour immunity. Although the historical background and preclinical data are promising, systemic therapy with TNF in human cancer has proved highly toxic and is inactive against all tumour types so far tested. Local therapy, particularly isolated limb perfusion, has resulted in complete and long lasting tumour regressions with necrotic activity confined solely to the tumour vascular bed. However, in several animal models, TNF contributes to malignant progression and there is evidence that TNF may have autocrine or paracrine actions in human ovarian cancer.     

Deficient spontaneous in vitro apoptosis and increased tmTNF reverse signaling-induced apoptosis of monocytes predict suboptimal therapeutic response of rheumatoid arthritis to TNF inhibition

Introduction

In vitro apoptosis of peripheral monocytes in rheumatoid arthritis (RA) is disturbed and influenced by cytokine production and transmembrane TNF (tmTNF) reverse signaling. The goal of the study was the analysis of the predictive value of the rate of in vitro apoptosis for the therapeutic response to anti-TNF treatment.

Methods

Spontaneous and tmTNF reverse signaling-induced apoptosis were determined in vitro in monocytes from 20 RA patients prior to initiation of therapeutic TNF inhibition with etanercept, and the subsequent clinical response was monitored.

Results

Spontaneous in vitro apoptosis was significantly reduced in RA patients compared to controls. Deficiency in spontaneous apoptosis was associated with an insufficient therapeutic response according to the European League Against Rheumatism (EULAR) response criteria and less reduction of the disease activity determined by disease activity score (DAS) 28. High susceptibility to reverse signaling-induced apoptosis was also associated with less efficient reduction in the DAS28. Of note, a strong negative correlation between the two apoptotic parameters was discernible, possibly indicative of two pathogenetically relevant processes counter-regulating each other.tmTNF reverse signaling induced in vitro production of soluble IL1-RI and IL-1RII only in monocytes not deficient in spontaneous apoptosis, and the levels of soluble IL1-RII were found to be predictive of a good clinical response to Etanercept.

Conclusion

Although tmTNF reverse signaling is able to induce apoptosis of RA monocytes in vitro, this process appears to occur in vitro preferentially in patients with suboptimal therapeutic response. Resistance to spontaneous in vitro apoptosis, in contrast, is a predictor of insufficient response to treatment.     

PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?

Dysregulated signal transduction in innate and adaptive immune cells is known to be associated with the development of various autoimmune and inflammatory diseases. Consequently, targeting intracellular signalling of the pro-inflammatory cytokine network heralds hope for the next generation of anti-inflammatory drugs. Phosphoinositide 3-kinases (PI3Ks) generate lipid-based second messengers that control an array of intracellular signalling pathways that are known to have important roles in leukocytes. In light of the recent progress in the development of selective PI3K inhibitors, and the beneficial effects of these inhibitors in models of acute and chronic inflammatory disorders, we discuss the therapeutic potential of blocking PI3K isoforms for the treatment of rheumatoid arthritis and other immune-mediated diseases.     

Differences in reactivation of tuberculosis induced from anti-TNF treatments are based on bioavailability in granulomatous tissue

The immune response to Mycobacterium tuberculosis (Mtb) infection is complex. Experimental evidence has revealed that tumor necrosis factor (TNF) plays a major role in host defense against Mtb in both active and latent phases of infection. TNF-neutralizing drugs used to treat inflammatory disorders have been reported to increase the risk of tuberculosis (TB), in accordance with animal studies. The present study takes a computational approach toward characterizing the role of TNF in protection against the tubercle bacillus in both active and latent infection. We extend our previous mathematical models to investigate the roles and production of soluble (sTNF) and transmembrane TNF (tmTNF). We analyze effects of anti-TNF therapy in virtual clinical trials (VCTs) by simulating two of the most commonly used therapies, anti-TNF antibody and TNF receptor fusion, predicting mechanisms that explain observed differences in TB reactivation rates. The major findings from this study are that bioavailability of TNF following anti-TNF therapy is the primary factor for causing reactivation of latent infection and that sTNF—even at very low levels—is essential for control of infection. Using a mathematical model, it is possible to distinguish mechanisms of action of the anti-TNF treatments and gain insights into the role of TNF in TB control and pathology. Our study suggests that a TNF-modulating agent could be developed that could balance the requirement for reduction of inflammation with the necessity to maintain resistance to infection and microbial diseases. Alternatively, the dose and timing of anti-TNF therapy could be modified. Anti-TNF therapy will likely lead to numerous incidents of primary TB if used in areas where exposure is likely.     

Lyn-dependent signaling regulates the innate immune response by controlling dendritic cell activation of NK cells

The innate immune response is a first line of defense against invading pathogens; however, the magnitude of this response must be tightly regulated, as hyper- or suboptimal responses can be detrimental to the host. Systemic inflammation resulting from bacterial infection can lead to sepsis, which remains a serious problem with high mortality rates. Lyn tyrosine kinase plays a key role in adaptive immunity, although its role in innate immunity remains unclear. In this study, we show that Lyn gain-of-function (Lyn(up/up)) mice display enhanced sensitivity to endotoxin and succumb to upregulated proinflammatory cytokine production at a dose well tolerated by control animals. Endotoxin sensitivity in Lyn(up/up) mice depends on dendritic cells (DCs) and NK cells and occurs though a mechanism involving increased maturation and activation of the DC compartment, leading to elevated production of IFN-γ by NK cells. We further show that modulation of endotoxin-induced signal transduction in DCs by Lyn involves the phosphatases Src homology 2 domain-containing phosphatase-1 and SHIP-1. Collectively, we demonstrate that Lyn regulates DC physiology such that alterations in Lyn-dependent signaling have profound effects on the nature and magnitude of inflammatory responses. Our studies highlight how perturbations in signaling pathways controlling DC/NK cell-regulated responses to microbial products can profoundly affect the magnitude of innate immune responses.     

Suppression of the effector phase of inflammatory arthritis by double-stranded RNA is mediated by type I IFNs

Innate immune receptors that recognize nucleic acids, such as TLRs and RNA helicases, are potent activators of innate immunity that have been implicated in the induction and exacerbation of autoimmunity and inflammatory arthritis. Polyriboinosine-polyribocytidylic acid sodium salt (poly(IC)) is a mimic of dsRNA and viral infection that activates TLR3 and the RNA helicases retinoic acid-induced gene-1 and melanoma differentiation-associated gene-5, and strongly induces type I IFN production. We analyzed the effects of systemic delivery of poly(IC) on the inflammatory effector phase of arthritis using the collagen Ab-induced and KRN TCR-transgenic mouse serum-induced models of immune complex-mediated experimental arthritis. Surprisingly, poly(IC) suppressed arthritis, and suppression was dependent on type I IFNs that inhibited synovial cell proliferation and inflammatory cytokine production. Administration of exogenous type I IFNs was sufficient to suppress arthritis. These results suggest a regulatory role for innate immune receptors for dsRNA in modulating inflammatory arthritis and provide additional support for an anti-inflammatory function of type I IFNs in arthritis that directly contrasts with a pathogenic role in promoting autoimmunity in systemic lupus.