Tuning IL-2 signaling by ADP-ribosylation of CD25.

Control of immunologic tolerance and homeostasis rely on Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5, and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells.

Selection of nanobodies that block the enzymatic and cytotoxic activities of the binary Clostridium difficile toxin CDT.

The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease.

Extracellular NAD(+): a danger signal hindering regulatory T cells.

Endogenous danger signals released during cell damage contribute to alert the immune system. Typically, their release results in the activation and maturation of innate immune cells, and the production of pro-inflammatory cytokines. In addition, extracellular NAD(+) stimulates immune responses by hindering regulatory T cells (Tregs), and could, therefore, represent the prototype of a new category of danger signals.

Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (T reg cells) play a major role in the control of immune responses but the factors controlling their homeostasis and function remain poorly characterized. Nicotinamide adenine dinucleotide (NAD(+)) released during cell damage or inflammation results in ART2.2-mediated ADP-ribosylation of the cytolytic P2X7 receptor on T cells. We show that T reg cells express the ART2.2 enzyme and high levels of P2X7 and that T reg cells can be depleted by intravenous injection of NAD(+). Moreover, lower T reg cell numbers are found in mice deficient for the NAD-hydrolase CD38 than in wild-type, P2X7-deficient, or ART2-deficient mice, indicating a role for extracellular NAD(+) in T reg cell homeostasis. Even routine cell preparation leads to release of NAD(+) in sufficient quantities to profoundly affect T reg cell viability, phenotype, and function. We demonstrate that T reg cells can be protected from the deleterious effects of NAD(+) by an inhibitory ART2.2-specific single domain antibody. Furthermore, selective depletion of T reg cells by systemic administration of NAD(+) can be used to promote an antitumor response in several mouse tumor models. Collectively, our data demonstrate that NAD(+) influences survival, phenotype, and function of T reg cells and provide proof of principle that acting on the ART2-P2X7 pathway represents a new strategy to manipulate T reg cells in vivo.

Genetic control of IL-1 beta production and inflammatory response by the mouse Irm1 locus.

Genome-wide linkage analysis using single nucleotide polymorphism arrays was carried out in pedigrees of mice differing in the extent of acute inflammatory response (AIRmax or AIRmin). The AIR phenotype was determined by quantifying the number of infiltrating cells in the 24-h exudate induced by Biogel P-100 s.c. injection and by ex vivo IL-1beta production by leukocytes stimulated with LPS and ATP. We mapped the major inflammatory response modulator 1 locus on chromosome 7, at the 1-logarithm of odds (LOD) confidence interval from 116.75 to 139.75 Mb, linked to the number of infiltrating cells (LOD = 3.61) through the production of IL-1beta (LOD = 9.35). Of several interesting candidate genes mapping to the inflammatory response modulator 1 locus, 28 of these were differentially expressed in the bone marrow of AIRmax and AIRmin mice. These findings represent a step toward the identification of the genes underlying this complex phenotype.

Cutting edge: CD4-independent development of functional FoxP3+ regulatory T cells.

The CD4 coreceptor is mandatory for the differentiation and function of conventional MHC class II-restricted T cells, but little is known about its contribution in regulatory T cells (Tregs). We thus investigated the Treg compartment in mice lacking CD4. CD3+CD8-FoxP3+ cells were readily detected in the periphery of CD4(-/-) mice, where their percentages were even increased as compared with wild-type animals. These cells had a classical CD25+CD152+GITR+ Treg phenotype, were enriched in memory-type Tregs, and displayed a diversified TCR repertoire. Functionally, CD4(-/-) Tregs were equally as suppressive as CD4(+/+) Tregs in vitro as well as in vivo. Hence, the CD4 coreceptor is dispensable for the generation and function of FoxP3+ Tregs. Furthermore, CD3+CD8-FoxP3+ Tregs were also found to develop in the absence of both CD4 and MHC-II molecules, demonstrating that the generation of Tregs can occur independently of MHC-II recognition.

Differential regulation of P2X7 receptor activation by extracellular nicotinamide adenine dinucleotide and ecto-ADP-ribosyltransferases in murine macrophages and T cells.

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X(7) purinergic receptor (P2X(7)R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X(7)R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X(7)R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X(7)R function in murine macrophages. Coexpression of the cloned murine P2X(7)R with ART2.1 or ART2.2 in HEK293 cells verified that P2X(7)R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X(7)R. Rather, NAD potentiated ATP-dependent P2X(7)R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X(7)R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X(7)R channel opening in T cells, this P2X(7)R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X(7)R signaling in murine macrophages and also suggest that the cellular context in which P2X(7)R signaling occurs differs between myeloid and lymphoid leukocytes.

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

Basal and inducible expression of the thiol-sensitive ART2.1 ecto-ADP-ribosyltransferase in myeloid and lymphoid leukocytes.

ADP-ribosylation of cell surface proteins in mammalian cells is a post-translational modification by which ecto-ADP-ribosyltransferases (ARTs) transfer ADP-ribose from extracellular NAD to protein targets. The ART2 locus at murine chromosome 7 encompasses the tandem Art2a and Art2b genes that encode the distinct ART2.1 and ART2.2 proteins. Although both ecto-enzymes share 80% sequence identity, ART2.1 activity is uniquely regulated by an allosteric disulfide bond that is reducible in the presence of extracellular thiols, such as cysteine and glutathione, that accumulate in hypoxic and ischemic tissues. Previous studies have characterized the expression of ART2.1 and ART2.2 in murine T lymphocytes but not in other major classes of lymphoid and myeloid leukocytes. Here, we describe the expression of ART2.1 activity in a wide range of freshly isolated or tissue-cultured murine myeloid and lymphoid leukocytes. Spleen-derived macrophages, dendritic cells (DC), and B cells constitutively express ART2.1 as their predominant ART while spleen T cells express both ART2.1 and the thiol-independent ART2.2 isoform. Although bone-marrow-derived macrophages (BMDM) and dendritic cells (BMDC) constitutively express ART2.1 at low levels, it is markedly up-regulated when these cells are stimulated in vitro with IFNbeta or IFNgamma. ART2.1 expression and activity in splenic B cells is modestly up-regulated during incubation in vitro for 24 h, a condition that promotes B cell apoptosis. This increase in ART2.1 is attenuated by IL-4 (a B cell survival factor), but is not affected by IFNbeta/gamma, suggesting a possible induction of ART2.1 as an ancillary response to B cell apoptosis. In contrast, ART2.1 and ART2.2, which are highly expressed in freshly isolated splenic T cells, are markedly down-regulated when purified T cells are incubated in vitro for 12-24 h. Studies with the BW5147 mouse thymocyte line verified basal expression of ART2.1 and ART2.2, as in primary spleen T cells, and demonstrated that both isoforms can be up-regulated when T cells are maintained in the presence of IFNs. Comparison of the surface proteins which are ADP-ribosylated by ART2.1 in the different leukocyte subtypes indicated both shared and cell-specific proteins as ART2.1 substrates. The LFA-1 integrin, a major target for ART2.2 in T cells, is also ADP-ribosylated by the ART2.1 expressed in macrophages. Thus, ART2.1, in contrast to ART2.2, is expressed in a broad range of myeloid and lymphoid leukocytes. The thiol redox-sensitive nature of this ecto-enzyme suggests an involvement in purinergic signaling that occurs in the combined context of inflammation and hypoxia/ischemia.

Activation of the P2X7 ion channel by soluble and covalently bound ligands.

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7.

NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells.

Extracellular NAD(+) and ATP trigger the shedding of CD62L and the externalization of phosphatidylserine on murine T cells. These events depend on the P2X(7) ion channel. Although ATP acts as a soluble ligand to activate P2X(7), gating of P2X(7) by NAD(+) requires ecto-ADP-ribosyltransferase ART2.2-catalyzed transfer of the ADP-ribose moiety from NAD(+) onto Arg125 of P2X(7). Steady-state concentrations of NAD(+) and ATP in extracellular compartments are highly regulated and usually are well below the threshold required for activating P2X(7). The goal of this study was to identify possible endogenous sources of these nucleotides. We show that lysis of erythrocytes releases sufficient levels of NAD(+) and ATP to induce activation of P2X(7). Dilution of erythrocyte lysates or incubation of lysates at 37 degrees C revealed that signaling by ATP fades more rapidly than that by NAD(+). We further show that the routine preparation of primary lymph node and spleen cells induces the release of NAD(+) in sufficient concentrations for ART2.2 to ADP-ribosylate P2X(7), even at 4 degrees C. Gating of P2X(7) occurs when T cells are returned to 37 degrees C, rapidly inducing CD62L-shedding and PS-externalization by a substantial fraction of the cells. The "spontaneous" activation of P2X(7) during preparation of primary T cells could be prevented by i.v. injection of either the surrogate ART substrate etheno-NAD or ART2.2-inhibitory single domain Abs 10 min before sacrificing mice.

Characterisation of the R276A gain-of-function mutation in the ectodomain of murine P2X7.

The cytolytic P2X7 purinoceptor is widely expressed on leukocytes and has sparked interest because of its key role in the activation of the inflammasome, the release of the pro-inflammatory cytokine IL-1beta and cell death. We report here the functional characterisation of a R276A gain-of-function mutant analysed for its capacities to induce membrane depolarisation, calcium influx and opening of a large membrane pore permeable to YO-PRO-1. Our results highlight the particular sensitivity of R276A mutant to low micromolar adenosine triphosphate (ATP) concentrations, which possibly reflect an increased affinity for its ligands, and a slower closing kinetics of the receptor channel. Our findings support the notion that evolutionary pressures maintain the low sensitivity of P2X7 to ATP. We also believe that the R276A mutant described here may be useful for the generation of new animal models with exacerbated P2X7 functions that will serve to better characterise its role in inflammation and in immune responses.

Increase of monoamine oxidase-B activity in the brain of scrapie-infected hamsters.

In the present study, the purpose is to determine activities of monoamine oxidases (MAO) in the brain of 263K scrapie-infected hamsters during the development of this experimental prion disease. Indeed, MAO activity modifications which have already been related in aging and neurodegenerations is suspected to be involved in the neuron loss process by elevated hydrogen peroxide formation. Monoamine oxidase type A (MAO-A) and B (MAO-B) activities were followed in the brain at different stages of the disease. MAO-A activity did not change significantly during the evolution of the disease. However, concerning the MAO-B activity, a significant increase was observed from 50 days post-infection and through the course of the disease and reached 42.9+/-5.3% at its ultimate stage. Regarding these results, MAO-B could be a potential therapeutic target then we have performed a pre-clinical treatment with irreversible (Selegiline or L-deprenyl) or and reversible (MS-9510) MAO-B inhibitors used alone or in association with an anti-scrapie drug such as MS-8209, an amphotericin B derivative. Our results show that none of the MAO-B inhibitors used was able to delay the onset of the disease. Neither these MAO-B inhibitors nor R-NMDA inhibitors (MK-801) can enhance the effects of MS-8209. The present findings clearly indicate a significant increase of cerebral MAO-B activity in scrapie-infected hamsters. Furthermore, inhibitors of MAO-B do not have any curative or palliative effect on this experimental model indicating that the raise of this activity is probably more a consequence rather than a causal event of the neurodegenerative process.

ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site.

ADP-ribosylation is a post-translational modification regulating protein function in which amino acid-specific ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD onto specific target proteins. Attachment of the bulky ADP-ribose usually inactivates the target by sterically blocking its interaction with other proteins. P2X7, an ATP-gated ion channel with important roles in inflammation and cell death, in contrast, is activated by ADP-ribosylation. Here, we report the structural basis for this gating and present the first molecular model for the activation of a target protein by ADP-ribosylation. We demonstrate that the ecto-enzyme ART2.2 ADP-ribosylates P2X7 at arginine 125 in a prominent, cysteine-rich region at the interface of 2 receptor subunits. ADP-ribose shares an adenine-ribonucleotide moiety with ATP. Our results indicate that ADP-ribosylation of R125 positions this common chemical framework to fit into the nucleotide-binding site of P2X7 and thereby gates the channel.

Lipopolysaccharide, IFN-gamma, and IFN-beta induce expression of the thiol-sensitive ART2.1 Ecto-ADP-ribosyltransferase in murine macrophages.

Nicotinamide adenosine dinucleotide (NAD) can act as a modulator of multiple immune and inflammatory responses when released into extracellular compartments. These actions of extracellular NAD are largely mediated by a family of mammalian ecto-ADP-ribosyltransferases (ARTs) that covalently modify target extracellular or cell surface proteins by transferring ADP-ribose to arginine or cysteine residues. In this study, we report that bone marrow-derived macrophages (BMDM) from BALB/c mice lack constitutive expression of any of the six murine ecto-ART subtypes, but selectively up-regulate ART2.1 in response to multiple proinflammatory mediators including agonists for TLR and type I and type II IFN. Stimulation of BMDM with LPS, IFN-beta, or IFN-gamma induced high expression of ART2.1, but not ART2.2, as a GPI-anchored cell surface ectoenzyme. ART2.1 expression in response to LPS was potentiated by inhibition of ERK1/2 signaling, but inhibited by blockade of the NF-kappaB, PI3K, and JAK-STAT pathways or the presence of neutralizing anti-IFN-beta. The catalytic function of the induced cell surface ART2.1 was strictly dependent on the presence of extracellular thiol-reducing cofactors, suggesting that in vivo activity of ART2.1-expressing macrophages may be potentiated in hypoxic or ischemic compartments. Consistent with the mutated art2a gene in C57BL/6 mice, LPS- or IFN-stimulated BMDM from this strain lacked expression of cell surface ART2 activity in the presence or absence of extracellular thiol reductants. Collectively, these studies identify ART2.1 as a new candidate for linking autocrine/paracrine activation of inflammatory macrophages to the release of NAD, a critical intracellular metabolite.

NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo.

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of ADP-ribose groups from NAD(+) onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP receptor and is responsible for NAD(+)-induced T cell death (NICD). Yet, the origin of extracellular NAD(+) and the role of NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release of NAD(+) into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD(+) glycohydrolase, whereas no effect is observed in ART2-deficient animals. Intravenous injection of NAD(+) used to exacerbate NICD in vivo results in fast and dramatic ART2- and P2X7-dependent depletion of CD4+ and CD8+ T lymphocytes, which can affect up to 80% of peripheral T cells in CD38(-/-) mice. This affects mainly naive T cells as most cells surviving in vivo NAD+ treatment exhibit the phenotype of recently activated/memory cells. Consistently, treatment with NAD(+) abolishes primary Ab response to a T-dependent Ag in NICD-susceptible CD38(-/-) mice but has no effect on the secondary response when given several days after priming. Unexpectedly NAD+ treatment improves the response in their wild-type BALB/c counterparts. We propose that NAD(+) released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of resting cells, thus contributing to the dynamic regulation of T cell homeostasis.

Extracellular NAD and ATP: Partners in immune cell modulation.

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as "danger signals" to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response.

Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation.

Following their release from cells, ATP and NAD, the universal currencies of energy metabolism, function as extracellular signalling molecules. Mammalian cells express numerous purinoceptors, i.e., the nucleotide-gated P2X ion channels and the G-protein-coupled P2Y receptors. Signalling through purinoceptors is controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides. These enzymes include ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) and ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP, CD203 family). Investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize these proteins in their native conformation. This study reports the use of genetic immunization to generate such antibodies against P2X(1), P2X(4), P2X(7), ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6. Genetic immunization ensures expression of the native protein by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs). Such antibodies are especially useful for immunofluorescence and immunoprecipitation analyses, whereas antibodies against synthetic peptides usually function well only in Western-blot analyses. Here we illustrate the utility of the new antibodies to monitor the cell surface expression of and to purify some key players of purinergic signalling.

Genetic determinants of acute inflammation regulate Salmonella infection and modulate Slc11a1 gene (formerly Nramp1) effects in selected mouse lines.

Two lines of mice selected to produce maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactions (AIR) differ in their susceptibility to infection by Salmonella enterica serotype Typhimurium (S. Typhimurium). The LD(50) for AIRmax mice is 1000 times higher than that observed for AIRmin mice, and higher frequencies of Slc11a1 alleles (known to confer either resistance (R) or high susceptibility (S) to S. Typhimurium) were consistently found in AIRmax and AIRmin mouse lines, respectively. In order to evaluate the effect of the quantitative trait loci (QTL) segregated in AIRmax and AIRmin mice on Slc11a1 dependent susceptibility to S. Typhimurium, the R and S alleles were fixed in homozygosity in AIRmax and AIRmin backgrounds by genotype assisted breedings. These new lines were named AIRmax(RR), AIRmax(SS), AIRmin(RR), and AIRmin(SS). Acute inflammation of Slc11a1(RR) animals was more severe in comparison to their Slc11a1(SS) counterparts, implicating Slc11a1 (or other linked genes) in AIR regulation. The LD(50) of S. Typhimurium was 800-times higher for AIRmax(SS) than for AIRmin(SS), demonstrating that AIR QTL can act as modifiers of the Slc11a1(SS) susceptibility gene. Four microsatellite markers for S. Typhimurium susceptibility QTL described in other mouse lines showed specific allele fixation in AIRmax or AIRmin mice, suggesting that these chromosomal regions also segregate with inflammatory phenotypes.

Inverse genetic predisposition to colon versus lung carcinogenesis in mouse lines selected based on acute inflammatory responsiveness.

Mouse lines produced by bidirectional selection on the basis of maximum (AIRmax) or minimum (AIRmin) acute inflammatory reactions were examined for the development of chemically induced acute colitis and colon tumors and the development of lung tumors. AIRmax mice were more susceptible than AIRmin to acute colitis induced by ingestion of dextran sodium sulfate showing a 3-fold higher disease activity index and presenting an intense inflammatory infiltrate in the base of colon crypts as well as elevated expression of IL-1beta, TNFalpha, IFNgamma and IL-6 mRNA in colon tissue. AIRmax were also more susceptible than AIRmin to colon cancer induced by 2 or 7 weekly doses of 1,2-dimethylhydrazine (DMH), showing significantly higher numbers of colonic aberrant crypt foci (ACF) at 150 days after DMH treatment (P = 0.01) and significantly higher numbers of tumors affecting larger intestinal areas at 300-475 days. At the latter time point, however, multiple lung adenomas and large adenocarcinomas were found in AIRmin but not in AIRmax mice. Treatment of mice with nimesulide for 60 days beginning 24 h before the first of two DMH doses almost completely inhibited the appearance of ACF in both lines. Furthermore, ACF numbers and the degree of acute inflammation directly co-segregated in an F2 (AIRmax x AIRmin) intercross population. The results demonstrate that genetic determinants of the inflammatory response differentially influence susceptibility to colon and lung carcinogenesis in the AIRmax and AIRmin mouse model.