Regulation of T Cell Priming by Lymphoid Stroma

The priming of immune T cells by their interaction with dendritic cells (DCs) in lymph nodes (LN), one of the early events in productive adaptive immune responses, occurs on a scaffold of lymphoid stromal cells, which have largely been seen as support cells or sources of chemokines and homeostatic growth factors. Here we show that murine fibroblastic reticular cells (FRCs), isolated from LN of B6 mice, play a more direct role in the immune response by sensing and modulating T cell activation through their upregulation of inducible nitric oxide synthase (iNOS) in response to early T cell IFNγ production. Stromal iNOS, which only functions in very close proximity, attenuates responses to inflammatory DC immunization but not to other priming regimens and preferentially affects Th1 cells rather than Th2. The resultant nitric oxide production does not affect T cell-DC coupling or initial calcium signaling, but restricts homotypic T cell clustering, cell cycle progression, and proliferation. Stromal feedback inhibition thus provides basal attenuation of T cell responses, particularly those characterized by strong local inflammatory cues

Kinome-Wide Functional Genomics Screen Reveals a Novel Mechanism of TNFα-Induced Nuclear Accumulation of the HIF-1α Transcription Factor in Cancer Cells

Hypoxia-inducible factor-1 (HIF-1) and its most important subunit, HIF-1α, plays a central role in tumor progression by regulating genes involved in cancer cell survival, proliferation and metastasis. HIF-1α activity is associated with nuclear accumulation of the transcription factor and regulated by several mechanisms including modulation of protein stability and degradation. Among recent advances are the discoveries that inflammation-induced cytokines and growth factors affect protein accumulation of HIF-1α under normoxia conditions. TNFα, a major pro-inflammatory cytokine that promotes tumorigenesis is known as a stimulator of HIF-1α activity. To improve our understanding of TNFα-mediated regulation of HIF-1α nuclear accumulation we screened a kinase-specific siRNA library using a cell imaging–based HIF-1α-eGFP chimera reporter assay. Interestingly, this systematic analysis determined that depletion of kinases involved in conventional TNFα signaling (IKK/NFκB and JNK pathways) has no detrimental effect on HIF-1α accumulation. On the other hand, depletion of PRKAR2B, ADCK2, TRPM7, and TRIB2 significantly decreases the effect of TNFα on HIF-1α stability in osteosarcoma and prostate cancer cell lines. These newly discovered regulators conveyed their activity through a non-conventional RELB-depended NFκB signaling pathway and regulation of superoxide activity. Taken together our data allow us to conclude that TNFα uses a distinct and complex signaling mechanism to induce accumulation of HIF-1α in cancer cells. In summary, our results illuminate a novel mechanism through which cancer initiation and progression may be promoted by inflammatory cytokines, highlighting new potential avenues for fighting this disease

Foscan® (mTHPC) photosensitized macrophage activation: enhancement of phagocytosis, nitric oxide release and tumour necrosis factor-α-mediated cytolytic activity

Photodynamic activation of macrophage-like cells contributes to an effective outcome of photodynamic therapy (PDT) treatment. The possibility for an enhancement of macrophage activity by photosensitization with meta-tetra(hydroxyphenyl)chlorin (mTHPC) (1 μg ml−1) was studied in U937, monocyte cell line differentiated into macrophages (U937Φ cells). Phagocytic activity of U937Φ cells was evaluated by flow-cytometry monitoring of ingestion of fluorescein-labelled Escherichia coli particles. Increase in irradiation fluence up to 3.45 mJ cm−2 (corresponding irradiation time 15 s) resulted in significant increase in fluorescence signal (145%), while at higher light fluences the signal lowered down to the untreated control values. A light energy-dependent production of tumour necrosis factor-alpha (TNF-α) by photosensitized macrophages was further demonstrated using the L929 assay. The maximum TNF-α mediated cytolysis was observed at 28 mJ cm−2 and was 1.7-fold greater than that in control. In addition, we demonstrated a fluence-dependent increase in nitric oxide (NO) production by mTHPC-photosensitized macrophages. NO release increased gradually and reached a plateau after irradiation fluence of 6.9 mJ cm−2. Cytotoxicity measurements indicated that the observed manifestations of mTHPC-photosensitized macrophage activation took place under the sublethal light doses. The relevance of the present findings to clinical mTHPC-PDT is discussed. © 1999 Cancer Research Campaig

Arginine Metabolism by Macrophages Promotes Cardiac and Muscle Fibrosis in mdx Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD.We use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial effects of short-term treatments.Our findings demonstrate that arginine metabolism by arginase promotes fibrosis of muscle in muscular dystrophy and contributes to kyphosis. Our findings also show that long-term, dietary supplementation with arginine exacerbates fibrosis of dystrophic heart and muscles. Thus, commonly-practiced dietary supplementation with arginine by DMD patients has potential risk for increasing pathology when performed for long periods, despite reports of benefits acquired with short-term supplementation

Interferon Gamma Activated Macrophages Kill Mycobacteria by Nitric Oxide Induced Apoptosis

Mycobacterium tuberculosis is an intracellular pathogen of macrophages and escapes the macrophages' bactericidal effectors by interfering with phagosome-lysosome fusion. IFN-γ activation renders the macrophages capable of killing intracellular mycobacteria by overcoming the phagosome maturation block, nutrient deprivation and exposure to microbicidal effectors including nitric oxide (NO). While the importance about NO for the control of mycobacterial infection in murine macrophages is well documented, the underlying mechanism has not been revealed yet. In this study we show that IFN-γ induced apoptosis in mycobacteria-infected macrophages, which was strictly dependent on NO. Subsequently, NO-mediated apoptosis resulted in the killing of intracellular mycobacteria independent of autophagy. In fact, killing of mycobacteria was susceptible to the autophagy inhibitor 3-methyladenine (3-MA). However, 3-MA also suppressed NO production, which is an important off-target effect to be considered in autophagy studies using 3-MA. Inhibition of caspase 3/7 activation, as well as NO production, abolished apoptosis and elimination of mycobacteria by IFN-γ activated macrophages. In line with the finding that drug-induced apoptosis kills intracellular mycobacteria in the absence of NO, we identified NO-mediated apoptosis as a new defense mechanism of activated macrophages against M. tuberculosis

Age-Related Alteration of Arginase Activity Impacts on Severity of Leishmaniasis

It is well documented that ageing alters many aspects of immune responses; however, a causal relation between impaired immune functions in ageing individuals and the response to infection has not been established. Experimental leishmaniasis is an excellent model to analyse protective and pathological immune responses. Leishmania parasites are obligate intracellular pathogens and invade mainly macrophages, which have dual function: they can kill the parasites or promote their growth. We have recently shown that arginase, an enzyme induced in infected macrophages, is a key factor for parasite survival. Here, we show that ageing reduces the expression levels of arginase in macrophages, resulting in more efficient control of parasite growth. Our results suggest that age-related differences in the metabolism of arginase in macrophages might contribute to the higher susceptibility of children to leishmaniasis

Evasion by Stealth: Inefficient Immune Activation Underlies Poor T Cell Response and Severe Disease in SARS-CoV-Infected Mice

Severe Acute Respiratory Syndrome caused substantial morbidity and mortality during the 2002–2003 epidemic. Many of the features of the human disease are duplicated in BALB/c mice infected with a mouse-adapted version of the virus (MA15), which develop respiratory disease with high morbidity and mortality. Here, we show that severe disease is correlated with slow kinetics of virus clearance and delayed activation and transit of respiratory dendritic cells (rDC) to the draining lymph nodes (DLN) with a consequent deficient virus-specific T cell response. All of these defects are corrected when mice are treated with liposomes containing clodronate, which deplete alveolar macrophages (AM). Inhibitory AMs are believed to prevent the development of immune responses to environmental antigens and allergic responses by interacting with lung dendritic cells and T cells. The inhibitory effects of AM can also be nullified if mice or AMs are pretreated with poly I:C, which directly activate AMs and rDCs through toll-like receptors 3 (TLR3). Further, adoptive transfer of activated but not resting bone marrow–derived dendritic cells (BMDC) protect mice from lethal MA15 infection. These results may be relevant for SARS in humans, which is also characterized by prolonged virus persistence and delayed development of a SARS-CoV-specific immune response in individuals with severe disease

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection

L-arginine Supplementation Improves Responses to Injury and Inflammation in Dextran Sulfate Sodium Colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y+ cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS−/− mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity