Tomas Leanderson

Type I IFN Signaling Is Crucial for Host Resistance against Different Species of Pathogenic Bacteria

It is known that host cells can produce type I IFNs (IFN-αβ) after exposure to conserved bacterial products, but the functional consequences of such responses on the outcome of bacterial infections are incompletely understood. We show in this study that IFN-αβ signaling is crucial for host defenses against different bacteria, including group B streptococci (GBS), pneumococci, and Escherichia coli. In response to GBS challenge, most mice lacking either the IFN-αβR or IFN-β died from unrestrained bacteremia, whereas all wild-type controls survived. The effect of IFN-αβR deficiency was marked, with mortality surpassing that seen in IFN-γR-deficient mice. Animals lacking both IFN-αβR and IFN-γR displayed additive lethality, suggesting that the two IFN types have complementary and nonredundant roles in host defenses. Increased production of IFN-αβ was detected in macrophages after exposure to GBS. Moreover, in the absence of IFN-αβ signaling, a marked reduction in macrophage production of IFN-γ, NO, and TNF-α was observed after stimulation with live bacteria or with purified LPS. Collectively, our data document a novel, fundamental function of IFN-αβ in boosting macrophage responses and host resistance against bacterial pathogens. These data may be useful to devise alternative strategies to treat bacterial infections.

Identification of human S100A9 as a novel target for treatment of autoimmune disease via binding to quinoline-3-carboxamides.

Despite more than 25 years of research, the molecular targets of quinoline-3-carboxamides have been elusive although these compounds are currently in Phase II and III development for treatment of autoimmune/inflammatory diseases in humans. Using photoaffinity cross-linking of a radioactively labelled quinoline-3-carboxamide compound, we could determine a direct association between human S100A9 and quinoline-3-carboxamides. This interaction was strictly dependent on both Zn++ and Ca++. We also show that S100A9 in the presence of Zn++ and Ca++ is an efficient ligand of receptor for advanced glycation end products (RAGE) and also an endogenous Toll ligand in that it shows a highly specific interaction with TLR4/MD2. Both these interactions are inhibited by quinoline-3-carboxamides. A clear structure-activity relationship (SAR) emerged with regard to the binding of quinoline-3-carboxamides to S100A9, as well as these compounds potency to inhibit interactions with RAGE or TLR4/MD2. The same SAR was observed when the compounds ability to inhibit acute experimental autoimmune encephalomyelitis in mice in vivo was analysed. Quinoline-3-carboxamides would also inhibit TNFα release in a S100A9-dependent model in vivo, as would antibodies raised against the quinoline-3-carboxamide–binding domain of S100A9. Thus, S100A9 appears to be a focal molecule in the control of autoimmune disease via its interactions with proinflammatory mediators. The specific binding of quinoline-3-carboxamides to S100A9 explains the immunomodulatory activity of this class of compounds and defines S100A9 as a novel target for treatment of human autoimmune diseases.

IFN-beta Gene Deletion Leads to Augmented and Chronic Demyelinating Experimental Autoimmune Encephalomyelitis.

Since the basic mechanisms behind the beneficial effects of IFN-β in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-β gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN-β knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells to proliferate or produce IFN-γ in response to recall Ag. Consequently, we addressed the effect of IFN-β on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN-β KO mice acquired a higher incidence and augmented EAE regardless of the source of T cells. This shows that the anti-inflammatory effect of endogenous IFN-β is predominantly exerted on the effector phase of the disease. Histopathological investigations of CNS in the effector phase revealed an extensive microglia activation and TNF-α production in IFN-β KO mice; this was virtually absent in wt littermates. This coincided with an increase in effector functions of T cells in IFN-β KO mice, as measured by IFN-γ and IL-4 production. We suggest that lack of endogenous IFN-β in CNS leads to augmented microglia activation, resulting in a sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits.

Interferon-β is required for interferon-α production in mouse fibroblasts

The type I interferons — interferon-a (IFN-a) and interferon-b (IFN-b) — are critical for protection against viruses during the acute stage of viral infection [1,2]. Furthermore, type I interferons have been implicated as important mediators in the regulation of lymphocyte development [3], immune responses [4,5] and the maintenance of immunological memory of cytotoxic T cells [6,7]. The different IFN-a subtypes are encoded by 12 genes in the mouse [8] whereas IFN-b is encoded for by only one gene [9]. IFN-a and IFN-b have a high degree of sequence homology and are thought to interact with the same surface receptor on target cells [10,11]. As an approach to analysing the different biological functions of IFN-a and IFN-b, we have generated a mouse strain with an inactivated IFN-b gene. We report here that embryonic fibroblasts from such mice produce neither IFN-b nor IFN-a upon Sendai virus infection, whereas the production of IFN-a by leukocytes from the same strain of mice is intact. IFN-a production in embryonic fibroblasts from IFN-b–/– mice could be rescued by ‘priming’ the cells using exogenous IFN-b. These results imply a unique role for IFN-b in the induction of type I interferons in peripheral tissues.

Expansion, Selection and Mutation of Antigen-Specific B Cells in Germinal Centers

There are many points of decision in the life of a B cell the outcome of which is a matter of life or death. In other words, B cells, like the rest of the immune system, are constantly under selective pressure from the outside world. In most situations a B cell passes a selective barrier due to the properties exhibited by its receptor for antigen, cell surface immunoglobulin (sig). It has the capacity to influence the result of this selection test by intemal manipulation of its sIg as well as other cell surface molecules. For instance it can change the affinity of its sIg receptor via the mechanism of somatic mutation; it can enhance signalling via sIg by alteration in the level and/or coupling of accessory molecules (Hombach et al. 1990, Venkituraman et al. 1991); and it might bypass the selection process altogether by the use of other surface molecules such as CD40 (Liu et al. 1989, Banchereau et al. 1991) and regulation of oncogene products (e.g. bc!-2; McDonnel et al. 1989, Nunez et al. 1991).

S100A9 Interaction with TLR4 Promotes Tumor Growth

By breeding TRAMP mice with S100A9 knock-out (S100A9−/−) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9−/− and TLR4−/−, but not in RAGE−/− animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b+ cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

Inhibition of the development of chronic experimental autoimmune encephalomyelitis by laquinimod (ABR-215062) in IFN-β k.o. and wild type mice

Laquinimod is a novel oral immunomodulatory substance, which is currently developed for the treatment of multiple sclerosis (MS). The ability of laquinimod to inhibit disease development was investigated in chronic experimental autoimmune encephalomyelitis (chEAE) in IFN-beta k.o. mice and wild type mice. Laquinimod was shown to inhibit both disease development and histopathological changes in the CNS. Furthermore, laquinimod was found to be independent of endogenous IFN-beta for its effect in chEAE. When laquinimod was combined with exogenous IFN-beta, a synergistic disease inhibitory effect was seen. These findings using laquinimod in preclinical disease models for MS emphasize the potential of laquinimod in the future treatment of MS also in patients that do not respond to IFN-beta monotherapy. Furthermore, the results indicate that laquinimod may favourably be combined with IFN-beta.

Somatic Mutation of Immunoglobulin V Genes in Vitro

The molecular mechanism behind affinity maturation is the introduction of point mutations in immunoglobulin (Ig) V genes, followed by the selective proliferation of B cells expressing mutants with increased affinity for antigen. An in vitro culture system was developed in which somatic hypermutation of Ig V genes was sustained in primed B cells. Cognate T cell help and cross-linking of the surface Ig were required, whereas the addition of lipopolysaccharide or a CD40 ligand to drive proliferation was insufficient. This system should facilitate understanding of the molecular and cellular mechanisms that regulate somatic mutation and B cell selection.

Induction of NFκB responses by the S100A9 protein is TLR4-dependent.

Interactions between danger‐associated molecular patterns (DAMP) and pathogen‐associated molecular patterns (PAMP) and pattern recognition receptors such as Toll‐like receptors (TLRs) are critical for the regulation of the inflammatory process via activation of nuclear factor‐κB (NF‐κB) and cytokine secretion. In this report, we investigated the capacity of lipopolysaccharide (LPS) ‐free S100A9 (DAMP) protein to activate human and mouse cells compared with lipoprotein‐free LPS (PAMP). First, we showed that LPS and S100A9 were able to increase NF‐κB activity followed by increased cytokine and nitric oxide (NO) secretion both in human THP‐1 cells and in mouse bone marrow‐derived dendritic cells. Surprisingly, although S100A9 triggered a weaker cytokine response than LPS, we found that S100A9 more potently induced IκBα degradation and hence NF‐κB activation. Both the S100A9‐induced response and the LPS‐induced response were completely absent in TLR4 knockout mice, whereas it was only slightly affected in RAGE knockout mice. Also, we showed that LPS and S100A9 NF‐κB induction were strongly reduced in the presence of specific inhibitors of TLR‐signalling. Chloroquine reduced S100A9 but not LPS signalling, indicating that S100A9 may need to be internalized to be fully active as a TLR4 inducer. This was confirmed using A488‐labelled S100A9 that was internalized in THP‐1 cells, showing a raise in fluorescence after 30 min at 37°. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S100A9 are TLR4 agonists. Importantly, S100A9 induced stronger NF‐κB activation albeit weaker cytokine secretion than LPS, suggesting that S100A9 and LPS activated NF‐κB in a qualitatively distinct manner.

Antioxidant metabolism induced by quinic acid. increased urinary excretion of tryptophan and nicotinamide

For over 50 years, hippuric/quinic acids were believed to have no biological efficacy. Here data are presented to support the hypothesis that quinic acid is not responsible for any efficacy, but rather that quinic acid nutritionally supports the synthesis of tryptophan and nicotinamide in the gastrointestinal (GI) tract, and that this in turn leads to DNA repair enhancement and NF‐kB inhibition via increased nicotinamide and tryptophan production.