Assessment of chloroquine as a modulator of immune activation to improve CD4 recovery in immune nonresponding HIV-infected patients receiving antiretroviral therapy.

OBJECTIVES: Chloroquine (CQ), an anti-inflammatory drug, inhibits Toll-like receptor (TLR) signalling in plasmacytoid dendritic cells (pDCs) and may be beneficial for HIV-infected patients in whom immune activation persists despite effective antiretroviral therapy (ART). The effect of CQ on CD4 T-cell recovery and immune activation in immune nonresponding patients receiving successful ART was therefore studied. METHODS: Nineteen adults on ART with CD4 counts ≤ 350 cells/µL and undetectable viral load (VL) orally received CQ at 250 mg/day for 24 weeks. Side effects, CD4 and CD8 T-cell counts, VL, T-cell activation, pDC proportion and plasma inflammatory markers were assessed at baseline, at 24 weeks, and at 12 weeks after CQ discontinuation (clinicaltrial.org registration #NCT02004314). RESULTS: CQ was well tolerated and all patients maintained an undetectable VL. The absolute CD4 and CD8 T-cell counts and their percentages, the pDC proportion, T-cell activation, D-dimer and C-reactive protein (CRP) plasma levels and the kynurenine/tryptophan ratio did not change with CQ treatment. Among nine cytokines/chemokines measured, only levels of interferon (IFN)-α2 were significantly increased by CQ treatment. CONCLUSIONS: CQ was well tolerated in patients with low CD4 T-cell counts despite long-term effective ART; however, 24 weeks of CQ treatment did not improved CD4 T-cell recovery, lymphoid and myeloid immune activation or inflammatory markers.

Soluble CD40-ligand (sCD40L, sCD154) plays an immunosuppressive role via regulatory T cell expansion in HIV infection.

CD40/CD40-ligand (CD40L) signalling is a key stimulatory pathway which triggers the tryptophan (Trp) catabolizing enzyme IDO in dendritic cells and is immunosuppressive in cancer. We reported IDO-induced Trp catabolism results in a T helper type 17 (Th17)/regulatory T cell (Treg ) imbalance, and favours microbial translocation in HIV chronic infection. Here we assessed the link between sCD40L, Tregs and IDO activity in HIV-infected patients with different clinical outcomes. Plasmatic sCD40L and inflammatory cytokines were assessed in anti-retroviral therapy (ART)-naive, ART-successfully treated (ST), elite controllers (EC) and healthy subjects (HS). Plasma levels of Trp and its metabolite Kynurenine (Kyn) were measured by isotope dilution tandem mass spectrometry and sCD14 was assessed by enzyme-linked immunosorbent assay (ELISA). IDO-mRNA expression was quantified by reverse transcription-polymerase chain reaction (RT-PCR). The in-vitro functional assay of sCD40L on Treg induction and T cell activation were assessed on peripheral blood mononuclear cells (PBMCs) from HS. sCD40L levels in ART-naive subjects were significantly higher compared to ST and HS, whereas EC showed only a minor increase. In ART-naive alone, sCD40L was correlated with T cell activation, IDO-mRNA expression and CD4 T cell depletion but not with viral load. sCD40L was correlated positively with IDO enzymatic activity (Kyn/Trp ratio), Treg frequency, plasma sCD14 and inflammatory soluble factors in all HIV-infected patients. In-vitro functional sCD40L stimulation induced Treg expansion and favoured Treg differentiation by reducing central memory and increasing terminal effector Treg proportion. sCD40L also increased T cell activation measured by co-expression of CD38/human leucocyte antigen D-related (HLA-DR). These results indicate that elevated sCD40L induces immunosuppression in HIV infection by mediating IDO-induced Trp catabolism and Treg expansion.

Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples.

BACKGROUND: Reactive oxygen species (ROS) are essential for sperm function. However, excessive ROS production can impair sperm function and might be a factor contributing to male infertility. METHODS: We investigated the levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) as well as lipid peroxidation, as represented by thiobarbituric acid reactive species (TBARS), in blood and seminal plasma of 38 normozoospermic males from infertile couples (NSI-males), compared with that of 17 fertile volunteers (FV-males). RESULTS: TBARS levels in blood and seminal plasma were higher in NSI-males than in FV-males (P < 0.0002, P < 0.0003, respectively), as were AA levels (P < 0.0003, P < 0.00004, respectively). On the contrary, the blood and seminal plasma levels of DHA were lower in NSI-males than in FV-males (P < 0.02 and P < 0.05, respectively). The AA/DHA ratios in blood and seminal plasma were higher in NSI-males than in FV-males (P < 0.003, P < 0.0007, respectively). Significant correlations between seminal and blood plasma levels of TBARS (P < 0.0001, r = 0.548), AA (P < 0.0001, r = 0.571) and DHA (P < 0.0001, r = 0.506) were found. CONCLUSIONS: Our data provide new insight into lipid metabolism in male infertility and indicate that systemic oxidative stress resulting in increased lipid peroxidation and an altered fatty acid profile may be, at least in part, responsible for infertility even in normozoospermic males.

Novel pharmaceutical approaches for treating patients with cystic fibrosis.

Before the cloning of the CFTR gene in 1989, there were relatively few treatment options for the many phenotypes associated with cystic fibrosis (CF). The advancement of research in areas such as immunology, molecular biology and pharmacology have provided new insights into the mechanism and evolution of CF. More than 40 systematic clinical trials evaluating new therapies for CF are presently registered with the NIH. A great deal of effort is focused on the main cause of mortality: chronic and persistent lung infections. Intestinal malabsorption, pancreatic insufficiency, reduced bone mineral density and reproductive abnormalities are other manifestations of this disease that have been targeted by innovated treatments which are giving renewed hope to CF patients and their families. The following review is a summary of the novel pharmaceutical approaches for the treatment of cystic fibrosis aimed at improving both the quality and the longevity of the lives of patients afflicted with this devastating disease.

Toll-like receptors, cytokines and the immunotherapeutics of asthma.

Asthma is a complex disease caused by a poorly characterized set of genetic and environmental factors whose pathology is a result of immune dysregulation. Toll-like receptors are pathogen associated molecular pattern receptors expressed by many airway and pulmonary tissues as well as cells of the innate and adaptive immune system. Ligation of toll-like receptors can lead to a change in the expression levels of multiple inflammatory and anti-inflammatory mediators which are involved in the pathogenesis of asthma. These ligands and their receptors are therefore prime candidates in the search for immunotherapeutic treatments of asthma. The use of murine models of allergic asthma as tools for the genetic dissection of this disease should allow the molecular mechanisms underlying asthma to be identified and possibly used as further immunotherapeutic targets.

TLR7 ligand prevents allergen-induced airway hyperresponsiveness and eosinophilia in allergic asthma by a MYD88-dependent and MK2-independent pathway.

Asthma is one of the leading causes of childhood hospitalization, and its incidence is on the rise throughout the world. Currently, the standard treatment for asthma is the use of corticosteroids to try to suppress the inflammatory reaction taking place in the bronchial tree. Using a murine model of atopic allergic asthma employing a methacholine-hyperresponsive (A/J) as well as a hyporesponsive (C57BL/6) strain of mice sensitized and challenged with ovalbumin, we show that treatment with a synthetic Toll-like receptor 7 (TLR7) ligand (S-28463, a member of the imidazoquinoline family) prevents development of the asthmatic phenotype. Treatment with S-28463 resulted in a reduction of airway resistance and elastance following ovalbumin sensitization and challenge. This was accompanied by a dramatic reduction in infiltration of leukocytes, especially eosinophils, into the lungs of both C57BL/6 and A/J mice following OVA challenge. Treatment with S-28463 also abolished both the elevation in serum IgE level as well as the induction of IL-4, IL-5, and IL-13 by OVA challenge. The protective effects of S-28463 were also observed in MK2 knockout, but not MYD88 knockout, mice. We did not observe a switch in cytokine profile from T(H)2 to T(H)1, as both IL-12p70 and IFN-gamma levels were reduced following S-28463 treatment. These results clearly demonstrate the anti-inflammatory effect of imidazoquinolines in an allergic asthma model as well as the clinical potential of TLR7 ligands in the treatment of allergic diseases.

Cystic fibrosis lung disease following infection with Pseudomonas aeruginosa in Cftr knockout mice using novel non-invasive direct pulmonary infection technique.

To better understand the mechanism of lung infection with Pseudomonas aeruginosa (P. aeruginosa), many techniques have been developed in order to establish lung infection in rodents. A model of chronic lung infection, using tracheotomy to inoculate the bacteria, has been extensively used in the cystic fibrosis (CF) mouse model of lung infection. The cystic fibrosis transmembrane channel (Cftr) knockout (KO) mice are smaller than normal mice and are more sensitive to housing and nutritional conditions, leading to small amounts of animals being available for experiments. Because of these characteristics, and because of the invasiveness of the infection procedure which we, and others, have been using to mimic the lung infection, we sought to find an alternative way to study the inflammatory response during lung P. aeruginosa infection. The technique we describe here consists of the injection of bacterial beads directly into the lungs through the mouth without the need of any tracheal incisions. This technique of direct pulmonary delivery enables much faster infection of the animals compared with the intratracheal technique previously used. The use of this less invasive technique allows the exclusion of the surgery-related inflammation. Our results show that, using the direct pulmonary delivery technique, the KO mice were more susceptible to P. aeruginosa lung infection compared with their wild-type (WT) controls, as shown by their increased weight loss, higher bacterial burden and more elevated polymorphonuclear (PMN) alveolar cell recruitment into the lungs. These differences are consistent with the pathological profiles observed in CF patients infected with P. aeruginosa. Overall, this method simplifies the infection procedure in terms of its duration and invasiveness, and improves the survival rate of the KO mice when compared with the previously used intratracheal procedure.

Role of host phosphotyrosine phosphatase SHP-1 in the development of murine leishmaniasis.

Activation of host phosphotyrosine phosphatase SHP-1 by Leishmania and its subsequent impact on tyrosine phosphorylation-based signaling cascades were shown to represent an important mechanism whereby this pathogen may alter host cell functions. Herein, we report that Leishmania-induced macrophage SHP-1 activity is necessary for its survival within phagocytes through the attenuation of nitric oxide-dependent and -independent microbicidal mechanisms. In vivo, Leishmania major infection, which footpad inflammation is mostly undetectable in SHP-1-deficient viable motheaten mice, was accompanied by increased inducible nitric oxide synthase and activation of neutrophils. These enhanced cellular activities were paralleled by a marked activation of signaling events usually negatively regulated by SHP-1. Overall, this study firmly establishes that modulation of the signaling terminator SHP-1 by Leishmania is essential for its installment and propagation.

Clearance of infection with Mycobacterium bovis BCG in mice is enhanced by treatment with S28463 (R-848), and its efficiency depends on expression of wild-type Nramp1 (resistance allele).

The mouse bcg host resistance gene is known to control the activation of host macrophages for killing of intracellular parasites like Leishmania donovani as well as intracellular bacteria, including Mycobacterium bovis BCG and Salmonella enterica serovar Typhimurium. The Nramp1 gene has been mapped to this locus and affects the efficiency of macrophage activation. It has been shown that imidazoquinoline compounds, including S28463, are able to improve the clearance of a number of intracellular pathogens such as herpes simplex virus 2, human papillomavirus, and Leishmania. The goal of this study was to determine whether S28463 is efficient against infection with another intracellular pathogen, M. bovis BCG, and to determine the molecular basis underlying this effect. To achieve this, B10A.Nramp1(r) and B10A.Nramp1(-/-) mice were infected with M. bovis BCG and treated with S28463. The bacterial content in the spleen from these mice was assayed by a colony-forming assay. In addition, in vitro experiments were performed using bone marrow-derived macrophage cell lines from these mice. These cells were treated with S28463 and/or gamma interferon (IFN-gamma), and nitric oxide (NO) production was measured. Our study was able to show that S28463 acts in synergy with IFN-gamma to increase the production of NO in vitro. We were also able to demonstrate that mice that carried the resistant allele of the Nramp1 gene and were infected with M. bovis BCG responded to treatment with S28463, resulting in a decreased bacterial load after 2 weeks of treatment. Mice that do not express the Nramp1 gene responded only to a very large dose of S28463, and the response was not as efficient as that observed in mice carrying a wild-type Nramp1 allele. Our data provide evidence for the potential of S28463 as an immunomodulator that may be helpful in designing efficient strategies to improve host defense against mycobacterial infection.

Natural resistance to intracellular pathogens: modulation of macrophage signal transduction related to the expression of the Bcg locus.

In mice, the Bcg/Nramp1 gene of the chromosome 1 has been implicated in natural resistance or susceptibility to infection with several intramacrophage microorganisms. Functional studies of Bcg/Nramp1 congenic macrophages have shown that this gene has many pleiotropic effects on macrophage activation and function. Although a specific role of Bcg/Nramp1 in the control of pleiotropic effects has not been defined yet, several observations propose unifying hypothesis for its complex role: metal ion transport is the primary function of the Bcg/Nramp1 gene, the availability of metal ions as cofactors for many proteins results from this primary function and, in turn, the effect on signal transduction results from ion-regulated expression of cellular proteins and their functions. In the present study, we examined the possible alterations in signal transduction pathways related to different allelic expression of the Bcg locus in B10R (Bcgr/Nramp1s) and B10S (Bcgs/Nramp1r) macrophages. We have utilized 1-DE and 2-DE immunoblot analyses and investigated phosphorylation of proteins using either anti-phosphotyrosine antibody or antibodies recognizing specific phospho-forms of signaling proteins. In the basal state, B10R macrophages had a superior ability to phosphorylate p38 mitogen-activated protein kinase (MAPK) and manganese superoxide dismutase. B10S counterparts were characterized by increased phosphorylation of Erk1/Erk2 MAPKs. The activation of macrophages revealed higher phosphorylation of signal transducer and activator of transcription in response to interferon gamma and a rapid decline in the level of inhibitory kappa B-alpha protein induced by lipopolysaccharide in B10R macrophages compared to B10S. Altogether, our results demonstrate a link between allelic expression of the Bcg/Nramp1 gene and alterations in several macrophage signaling pathways, and support the hypothesis that the allelic expression of Bcg/Nramp1 may be functionally linked to resistance to infectious disease and, inversely, to autoimmune disease susceptibility.

Nramp1 gene affects selective early steps in macrophage-mediated anti-cryptococcal defense.

Cryptococcus neoformans is an opportunistic fungus responsible for severe and often recurrent meningoencephalitis in immunodepressed patients. Initial evidence suggests that C. neoformans is a facultative intracellular pathogen; however, the strategies by which C. neoformans undergoes survival and eventually proliferation have not been elucidated. We investigated the role of Nrampl gene in macrophage-mediated anti-cryptococcal defense. Using cell lines expressing the functional, mutated or knockout gene, it was established that Nramp1 (1) is not involved in the phagocytic event, (2) influences anti-cryptococcal activity in the early steps but not at later times, and (3) is unrelated to the biomolecular pathways through which C. neoformans impairs macrophage secretory response. Although the functional role of Nramp1 is still far from being elucidated, the present data add insight into its involvement in macrophage-mediated antimicrobial defense, particularly in the initial steps allowing C. neoformans growth inhibition.

Polymorphism in the 3'-untranslated region of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA.

Tumor necrosis factor alpha (TNFalpha) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3'-untranslated region (3'-UTR) of TNFalpha mRNA as this region plays a pivotal role in post-transcriptional control of TNFalpha production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFalpha mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFalpha. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3'-UTR of TNFalpha mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFalpha protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFalpha 3'-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFalpha mRNA, these results suggest that mutations in the ARE of TNFalpha mRNA decrease the production of TNFalpha protein in macrophages by hindering the binding of HuR to the ARE.

Mouse models of chronic lung infection with Pseudomonas aeruginosa: models for the study of cystic fibrosis.

The discovery of the CFTR gene in 1989 has lead to rapid progress in understanding the molecular basis of cystic fibrosis (CF) and the biological properties of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. However, more than 10 years later, recurrent lung infections with Pseudomonas aeruginosa, which lead to chronic lung disease and eventual respiratory failure, remain the major cause of morbidity and mortality among CF patients. A distinguishing feature of lung disease in CF is an exaggerated and persistent inflammatory response, characterized by the accumulation of excessive numbers of neutrophils and dysregulated cytokine production. The events leading to the establishment of lung infection with P. aeruginosa, especially the inflammatory and immunological events, and the relation between the CF defect and infection, remain largely undefined. Progress in this area has been hampered by the lack of a suitable animal model. An exciting achievement in the past few years has been the development of a number of variants of CFTR-deficient mice which exhibit defective cAMP-mediated Cl(-) conductance and have a range of clinical phenotypes from mild to severe. In parallel, a model of chronic P. aeruginosa lung infection has been established in genetically and immunologically well-defined inbred mouse strains which differ in susceptibility to this infection in the lung. BALB/c mice are resistant, while DBA/2 mice are extremely susceptible, with high mortality within 3 days of infection. C57BL/6 and A/J mice are relatively susceptible and experience low mortality. Furthermore, the bacterial load correlates with the magnitude and quality of the inflammatory response in the infected lungs of BALB/c and C57BL/6 mice. Although results of infection studies in CFTR-deficient mice have been variable, C57BL/6-Cftr(m1UNC)/Cftr(m1UNC) knockout mice compared to littermate control mice are highly susceptible to chronic P. aeruginosa infection in the lung. The availability of CFTR knockout mice and non-CF inbred mice differing in susceptibility to chronic P. aeruginosa infection offers useful tools for progress in understanding the genesis of chronic P. aeruginosa infection and the ensuing inflammation in the CF lung, as well as the relation between the CF defect and infection. Information generated from these studies will provide the rationale for the development of novel immunomodulatory measures capable of ameliorating or modulating the chronic inflammation associated with CF lung disease.

Airway surface liquid composition in mice.

Airway surface liquid (ASL) lines the conducting airways of the respiratory tract. We collected small samples of this liquid from the lower tracheae of anesthetized C57BL/6 mice and determined its ionic composition (in mM: 87.2 Na(+), 4.7 K(+), and 57.0 Cl(-)). Intravenous methacholine produced significant increases in the concentrations of Na(+), K(+), and Cl(-) within ASL. A limited analysis of liquid from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice revealed no significant differences compared with littermate controls; however, Pseudomonas aeruginosa infection led to an increase in the salt concentration of ASL in cftr(+/+) mice. Morphometric measurements of tracheal submucosal gland volume revealed significant differences between inbred mouse strains, corresponding to ease of ASL collection. We conclude that although submucosal glands may be responsible for the production of some ASL, the ionic composition of this liquid is actively regulated by the underlying epithelial cells.

Differential effector and secretory functions of microglial cell lines derived from BCG-resistant and -susceptible congenic mouse strains.

Using congenic strains of mice susceptible (bcg(s)) or resistant (bcg(r)) to BCG, murine microglial cell lines, RR4.R (BCG-resistant) and RR8.S (BCG-susceptible), were established in vitro. Comparative studies revealed that, although phagocytic to a similar extent, RR4.R cells were more active than RR8.S cells in terms of antimycobacterial activity. Interestingly, cells of resistant genotype secreted more nitric oxide, TNF-alpha and IL-12, but less IL-6, than susceptible cells, when stimulated with IFN-gamma alone or in combination with lipopolysaccharide. Nevertheless, no significant differences were observed between the two cell lines in terms of IL-1 beta or IL-10 secretion, or on assessment of cytokine production following exposure to a massive dose of lipopolysaccharide. Overall, these data provide the first evidence that resistant/susceptible genotype influences antimycobacterial activity, NO and cytokine production in microglial cells, the prototype of cerebral macrophages.

Attenuation of MHC class II expression in macrophages infected with Mycobacterium bovis bacillus Calmette-Guérin involves class II transactivator and depends on the Nramp1 gene.

The natural resistance associated macrophage protein 1 (Nramp1) gene determines the ability of murine macrophages to control infection with a group of intracellular pathogens, including Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). The expression of the resistant allele of the Nramp1 gene in murine macrophages is associated with a more efficient expression of several macrophage activation-associated genes, including class II MHC loci. In this study, we investigated the molecular mechanisms involved in IFN-gamma-induced MHC class II expression in three types of macrophages: those expressing a wild-type allele of the Nramp1 gene (B10R and 129/Mphi), those carrying a susceptible form of the Nramp1 gene (B10S), and those derived from 129-Nramp1-knockout mice (129/Nramp1-KO). Previously, we published results showing that Ia protein expression is significantly higher in the IFN-gamma-induced B10R macrophages, compared with its susceptible counterpart. In this paper, we also show that the higher expression of Ia protein in B10R cells is associated with higher I-Abeta mRNA expression, which correlates with a higher level of IFN-gamma-induced phosphorylation of the STAT1-alpha protein and subsequently with elevated expression of class II transactivator (CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the infection of macrophages with M. bovis BCG results in a down-regulation of CIITA mRNA expression and, consequently, in the inhibition of Ia induction. Therefore, our data explain, at least in part, the molecular mechanism involved in the inhibition of I-Abeta gene expression in M. bovis BCG-infected macrophages activated with IFN-gamma.

Innate resistance to experimental Trypanosoma congolense infection: differences in IL-10 synthesis by macrophage cell lines from resistant and susceptible inbred mice.

BALB/c and C57Bl/6 mice differ in resistance to T. congolense infections. We investigated the production of various cytokines (IL-10, IL-6, TNF-alpha and TGF-beta) by macrophages from these mice. Macrophage cell lines (BALB.BM cells) of BALB/c mice but not (ANA-I cells)of C57BL/6 mice constitutively produced IL-10. Challenge of these cells with trypanosomes induced the production of 50-100 times more IL-10 in BALB.BM cells than in ANA-1 cells. Pre-incubation of the cell lines with IFN-gamma. prior to the trypanosome challenge, further upregulated this IL-10 production in BALB.BM but not in ANA-1 cells. Primary cultures of bone marrow-derived macrophages (BMDM) from BALB/c mice also produced more IL-10 following challenge with IFN-gamma and opsonized trypanosomes than did the C57Bl/6 BMDM. Similarly after challenge with trypanosomes, BALB.BM and BALB/c BMDM produced significantly more IL-6 than did the analogous cells from the C57Bl/6 mice following such challenges. Higher steady state levels of TNF-alpha mRNA accumulated in ANA-1 cells than in BALB.BM cells following challenge with IFN-gamma and opsonized trypanosomes. Findings of this study for the first time indicate a differential regulation of cytokines (IL-10, IL-6 and TNF-alpha) in macrophages of mice that significantly differ in their susceptibility to infections with T. congolense.

Acquired resistance but not innate resistance to Mycobacterium bovis bacillus Calmette-Guérin is compromised by interleukin-12 ablation.

Interleukin-12 (IL-12) is one of the first cytokines produced by macrophages, key mediators of innate resistance, during the host's immune response to infections. Therefore, in this study we propose that IL-12 has an important role in the early phase of the immune response to Mycobacterium bovis BCG. IL-12 has been shown to enhance the maturation of protective Th1 cells and gamma interferon (IFN-gamma) production during mycobacterial infection. Therefore, it may play a crucial role during the immune phase of infection as well. To examine the role of IL-12 in both the innate and the immune phase of infection, we compared BCG-resistant mice, B10.A (Bcgr), to the susceptible congenic strain B10.A (Bcgs) following administration of a blocking monoclonal antibody to IL-12 (10F6). Anti-IL-12-treated susceptible animals exhibited a two- to threefold increase in spleen CFU by day 21. In contrast, anti-IL-12 treatment had little or no effect on the response of the genetically resistant animals to infection. The B10.A (Bcgr) but not the B10.A (Bcgs) mice had an increase in IFN-gamma mRNA relative to baseline levels as early as day 1 of infection irrespective of anti-IL-12 treatment. By day 14, B10.A (Bcgr) mice showed a decrease in IFN-gamma mRNA while the B10.A (Bcgs) mice showed a significant increase in IFN-gamma mRNA levels. Thus, during BCG infection, the B10.A (Bcgr) mice mount an early IFN-gamma response against BCG whereas the B10.A (Bcgs) mice have a delayed IFN-gamma response correlating with their genetic permissiveness expressed as an increased mycobacterial load by day 21. Overall, our data demonstrate that the inherent resistance of B10.A (Bcgr) mice to mycobacteria does not depend on optimal levels of IL-12 to maintain effective control of the bacteria, whereas IL-12 is important for the susceptible animals' response to BCG during the peak of infection.

Natural resistance to intracellular parasites: a study by two-dimensional gel electrophoresis coupled with multivariate analysis.

Natural resistance to Mycobacterium bovis bacillus Calmette-Guérin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two-dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophages transfected with Nramp1-ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele-carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn-superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl-2 according to the immunoblot analysis. When the splenic macrophages carrying the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.

Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor.

Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-kappaB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417-426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins-interleukin-10 (IL-10) and IL-6-also induced SLPI, while TNF and IL-1beta did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules-taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls-also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI's slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria.