Slc11a1 (formerly NRAMP1) gene modulates both acute inflammatory reactions and pristane-induced arthritis in mice

Mice selected for the maximum acute inflammatory reaction (AIRmax) are highly susceptible to pristane-induced arthritis (PIA), whereas mice selected for the minimum response (AIRmin) are resistant. These lines show distinct patterns of leukocyte infiltration and R and S allele frequency disequilibrium of the solute carrier family 11a member 1 (Slc11a1) gene. In order to study the interactions of the Slc11a1 R and S alleles with the inflammation modulating Quantitative Trait Loci (QTL) during PIA development, homozygous AIRmax(RR), AIRmax(SS), AIRmin(RR) and AIRmin(SS) lines were produced by genotype-assisted breedings. These mice received two intraperitoneal injections of 0.5 ml pristane at 60-day intervals, and the subsequent development of arthritis was assessed for 210 days. Cytokine-secreting cell profiles were investigated using enzyme-linked immunospot. Arthritis incidence in AIRmax(RR) mice reached 29%, whereas PIA incidence in AIRmax(SS) mice was 70% by day 180. AIRmin(RR) mice were resistant, whereas 13.3% of AIRmin(SS) mice became arthritic. The presence of the defective S allele also increased arthritis severity, although acute inflammation was higher in mice bearing the R allele. A predominant Th0/Th2-type response in Slc11a1(SS) mice was observed. These results indicate that Slc11a1 is a strong candidate for the QTL modulating acute inflammation and for PIA.     

Disruption of the Nramp1 (also known as Slc11a1) gene in Kupffer cells attenuates early-phase,warm ischemia-reperfusion injury in the mouse liver

As the natural resistance-associated macrophage protein 1 Nramp1 (also known as Slc11a1) modulates Kupffer cell (KC) activation, and KC are responsible for the early phase of warm ischemia/reperfusion (I/R) to the liver, we hypothesized that livers of Nramp1(-/-) mice will be protected from early-phase I/R injury compared with livers of Nramp1(+/+) mice. To test our hypothesis, we induced partial warm ischemia to the livers of Nramp1(+/+) and Nramp1(-/-) mice for 45 min of by clamping the hilum of the median and left lateral lobes, followed by 30 or 60 min of reperfusion. Plasma glutamate oxaloacetate transaminase (pGOT) activity and tumor necrosis factor alpha (TNF-alpha) levels were measured, and liver sections were stained for polymorphonuclear leukocyte (PMN) accumulation. After 45 min of ischemia and 30/60 min of reperfusion of Nramp1(+/+) and Nramp1(-/-) mice livers, we found significant increases in plasma pGOT activity and TNF-alpha levels in Nramp1(+/+) mice at 30 and 60 min of reperfusion, respectively, compared with sham controls and all Nramp1(-/-) mice. A significant accumulation of PMNs was also found in livers of Nramp1(+/+) mice at 60 min of reperfusion compared with all other groups. We have shown that disruption of the Nramp1 gene attenuates I/R injury to the mouse liver during the early phase of warm I/R injury. An increased understanding of the role played by Nramp1 is particularly important in the liver, as this organ is subjected to a wide variety of injuries during hemorrhagic shock, partial resections, and transplantation.     

Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance.

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.     

Stimulation of cell-mediated immunity by bestatin correlates with reduction of bacterial persistence in experimental chronic Salmonella typhimurium infection.

The effect of bestatin, a low-molecular-weight immunomodulating drug isolated from Streptomyces olivoreticuli, on Salmonella typhimurium infection was elaborated. Bestatin enhanced the delayed-type hypersensitivity reaction against S. typhimurium in a dose- and time-dependent manner. Parallel to the activation of delayed-type hypersensitivity reaction, bestatin reduced the amount of persistent bacteria in livers and spleens as well as the amount of necrotic foci found in these organs. This was shown when bestatin was given either prophylactically or therapeutically. The therapeutic effect of bestatin was even seen when the drug was given in the chronic phase of the infection, i.e., 6 days after inoculation of the animals with the infectious agent. No influence of bestatin, however, could be observed on the initial multiplication rate of S. typhimurium and concomitantly on the initial mortality rate of the infected mice. As bestatin has no direct antibiotic effect on S. typhimurium, it must be concluded that the therapeutic effects of the drug on chronic infection must be solely contributed to elevation of the host's own defense mechanisms.     

Isocitrate lyase (AceA) is required for Salmonella persistence but not for acute lethal infection in mice

Isocitrate lyase is required for fatty acid utilization via the glyoxylate shunt. Although isocitrate lyase is essential for Salmonella persistence during chronic infection, it is dispensable for acute lethal infection in mice. Substrate availability in the phagosome appears to evolve over time, with increasing fatty acid dependence during chronic infection.     

Identification of genetic loci controlling the characteristics and severity of brain and spinal cord lesions in experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is the principal genetically determined animal model for multiple sclerosis (MS), the major inflammatory disease of the central nervous system (CNS). Although genetics clearly play a role in susceptibility to MS, attempts to identify the underlying genes have been disappointing. Considerable variation exists between MS patients with regard to the severity of clinical signs, mechanism of demyelination, and location of CNS lesions, confounding the interpretation of genetic data. A mouse-human synteny mapping approach may allow the identification of candidate susceptibility loci for MS based on the location of EAE susceptibility loci. To date, 16 regions of the mouse genome have been identified that control susceptibility or clinical signs of EAE. In this work, we examined the genetic control of histopathological lesions of EAE in an F2 intercross population generated from the EAE susceptible SJL/J and EAE resistant B10.S/DvTe mouse strains. Composite interval mapping was used to identify 10 quantitative trait loci (QTL), including seven newly identified loci controlling the distribution and severity of CNS lesions associated with murine EAE. QTL on chromosome 10 control lesions in the brain, whereas QTL on chromosomes 3, 7, and 12 control lesions in the spinal cord. Furthermore, sexually dimorphic QTL on chromosomes 2, 9, and 11 control CNS lesions in females, whereas QTL on chromosomes 10, 11, 12, 16, and 19 control lesions in males. Our results suggest that the severity and location of CNS lesions in EAE are genetically controlled, and that the genetic component controlling the character and severity of the lesions can be influenced by sex.     

Identification and characterization of Cri1, a locus controlling mortality during Citrobacter rodentium infection in mice

Infection of inbred mouse strains with Citrobacter rodentium represents an ideal model to reveal the genetic factors controlling host resistance to noninvasive enteric bacterial pathogens. We have chosen a positional cloning approach to identify putative gene(s) that control the known difference in survival between resistant C57BL/6J and susceptible C3H/HeJ and C3H/HeOuJ mice. Our work has identified one major locus within proximal chromosome 15 that is responsible for the marked susceptibility of both C3H strains, and we formally exclude Tlr4 from control of survival to this pathogen. We have named this new host resistance locus Cri1 (Citrobacter rodentium infection 1). The Cri1 genetic interval currently spans ～16 Mb and it confers survival to the infection in a recessive manner. Transfer of the Cri1 locus from the surviving B6 mice into a congenic mouse with a C3Ou genetic background confirms its overall chromosomal localization and its highly significant effect on host survival. The C3Ou.B6-Cri1 mice thus produced have also enabled us to dissociate the control of mouse survival from the control of bacterial load early in the infection as well as from control of colonic hyperplasia.     

Divalent cation transport and susceptibility to infectious and autoimmune disease: continuation of the Ity/Lsh/Bcg/Nramp1/Slc11a1 gene story

Solute carrier family 11 member a1 (Slc11a1), formerly known as Nramp1/Ity/Lsh/Bcg, is a proton/divalent cation antiporter that regulates susceptibility to infectious and autoimmune disease. Here we review recent studies on (1) the role of Slc11a1 in iron metabolism and iron recycling in macrophages; (2) the use of mouse breeding and introgression of knockouts onto Slc11a1 congenic backgrounds for genes encoding the multiple pleiotropic functions associated with Slc11a1; and (3) associations/linkages of SLC11A1 with human disease and how these relate to functional promoter region polymorphisms.     

Genetic resistance of mice to Mycobacterium paratuberculosis is influenced by Slc11a1 at the early but not at the late stage of infection

We have recently described the development of a luminescent Mycobacterium paratuberculosis strain of bovine origin expressing the luxAB genes of Vibrio harveyi. With this luminescent isolate, fastidious and costly enumeration of CFU by plating them on agar can be replaced by easy and rapid luminometry. Here, we have reevaluated the effect of Slc11a1 (formerly Nramp1) polymorphism on susceptibility to M. paratuberculosis, using this luminometric method. A series of inbred mouse strains were infected intravenously with luminescent M. paratuberculosis S-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results indicate that, as for Mycobacterium avium subsp. avium, innate resistance to infection is genetically controlled by Slc11a1. In BALB/c, congenic BALB.B10-H2(b) (BALB/c background; H-2(b)), C57BL/6, and beige C57BL/6(bg/)(bg) mice (all Slc11a1(s)), bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection, whereas in DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (both Slc11a1(r)) and in (C57BL/6 x DBA/2)F(1) mice (Slc11a1(s/r)), the bacterial numbers had decreased more than 10-fold at 4 weeks postinfection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4 and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6(bg/)(bg) mice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice.     

Neural route of cerebral Listeria monocytogenes murine infection: role of immune response mechanisms in controlling bacterial neuroinvasion

The pathologic features of cerebral Listeria monocytogenes infection strongly suggest that besides hematogenous spread, bacteria might also spread via a neural route. We propose that after snout infection of recombination activating gene 1 (RAG-1)-deficient mice, L. monocytogenes spreads to the brain via a neural route. The neural route of invasion is suggested by (i) the immunostaining of L. monocytogenes in the trigeminal ganglia (TG) and brain stem but not in other areas of the brain; (ii) the kinetics of bacterial loads in snout, TG, and brain; and (iii) the increased resistance of mice infected with a plcB bacterial mutant (unable to spread from cell to cell). Gamma interferon (IFN-gamma) plays a protective role in neuroinvasion; inducible nitric oxide synthase (iNOS) accounts only partially for the protection, as shown by a comparison of the susceptibilities of IFN-gamma receptor (IFN-gamma R)-deficient, iNOS-deficient, and wild-type mice to snout infection with L. monocytogenes. The dramatically enhanced susceptibility of RAG-1-deficient, IFN-gamma R gene-deficient mice indicated the overall importance of innate immune cells in the release of protective levels of IFN-gamma. The source of IFN-gamma appeared to be NK cells, as shown by use of RAG-1-deficient, gamma-chain receptor gene-deficient mice; NK cells played a relevant protective role in neuroinvasion through a perforin-independent mechanism. In vitro evidence indicated that IFN-gamma can directly induce bacteriostatic mechanisms in neural tissue.     

Influence of macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) on Toxoplasma gondii infection in mice.

Functional studies have shown that the murine macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage priming/activation for antimicrobial activity via the tumour necrosis factor-alpha (TNF-alpha)-dependent production of reactive nitrogen intermediates. Since Toxoplasma gondii also parasitizes macrophages, is a stimulator of endogenous TNF-alpha release, and is sensitive to nitric oxide-mediated killing in activated macrophages, studies were carried out using chromosome 1 congenic mouse strains to determine whether Lsh influences T. gondii infection. Two interesting observations were made: (i) contrary to expectation, mice carrying the Lsh-resistant allele died earlier over the acute phase of infection than Lsh-susceptible mice; and (ii) Lsh-resistant mice which survived this acute phase of infection showed lower brain cyst numbers than the Lsh-susceptible mice. Whilst the latter occurred independently of route of inoculation (oral, intraperitoneal, or subcutaneous), the former was influenced both by the route of inoculation and the genetic background on which the Lsh-resistant allele had been isolated. Hence, following oral administration of 20 brain cysts of the RRA strain of T. gondii, mice carrying the Lsh-resistant allele on a B10 genetic background showed a significantly enhanced rate of mortality over the acute (first 8-12 days) phase of infection than B10 Lsh-susceptible mice. Although this acute phase of infection in B10 background mice was accompanied by an increase in serum TNF-alpha levels in both Lsh-resistant and -susceptible mouse strains, early mortality preceded the TNF-alpha peak, and administration of neutralizing rabbit anti-TNF-alpha did not significantly enhance survival. Hence, inflammatory mediators other than TNF-alpha appear to be responsible for the increased rate of acute mortality observed in resistant mice. Infection intraperitoneally led to delayed mortality in B10 mice, with the mean time to 50% mortality now being significantly longer in Lsh-resistant than in Lsh-susceptible mice. On a BALB genetic background, it was the i.p. route of infection which led to acute mortality and more rapid death in the Lsh-resistant strain. When a less virulent inoculum was used and mortality delayed, Lsh-susceptible mice died more rapidly, and i.p. administration of rabbit anti-TNF-alpha led to 100% mortality between days 8 and 10 of infection in both susceptible and resistant mouse strains, consistent with a crucial protective role for TNF-alpha during this phase of infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a peptide inducing experimental autoimmune uveoretinitis (EAU) in H-2Ak-carrying mice

When certain strains of mice bearing H-2A^k are immunized with the interphotoreceptor retinoid-binding protein (IRBP), EAU is induced. Thus far uveitogenic determinant(s) has not been determined in the H-2A^k mouse system. In addition it is hard to prepare purified IRBP. In the present study, to circumvent these problems we attempted to identify uveitogenic peptides derived from bovine IRBP in H-2A^k haplotype mice. Six peptides which had been selected according to the H-2A^k binding motif (Dxxxxxxxx[A, R, T]) were synthesized. We report here that all the peptides are immunogenic but only one peptide, K2, which consisted of IRBP201–216 residues, induces EAU in various mice carrying H-2A^k. Amino acid substitution of K2 revealed that the core region interacted with both H-2A^k and T cell antigen receptor (TCR). The amino acid sequence of the core region derived from bovine IRBP was identical to the corresponding region of mouse IRBP. In addition, K2 appeared to be a natural peptide antigen processed from bovine IRBP. Altogether, we concluded that K2 is one of the natural autoantigens involved in induction of EAU in H-2A^k mice.     

The role of Mac-1 (CD11b/CD18) in antigen-induced airway eosinophilia in mice

Mac-1 (CD11b/CD18) is an important adhesion molecule involved in the migration of leukocytes, cell signaling, and subsequent secretory responses. Its precise role in eosinophil recruitment and activation in vivo is not entirely clear. We wished to directly examine the role of Mac-1 in eosinophil migration in a murine model of allergic pulmonary inflammation. Briefly, wild-type (C57Bl/6) and Mac-1-deficient/knockout (Mac-1 KO) mice were intraperitoneally sensitized with ovalbumin (OVA) and alum (AlOH) on Days 0 and 14, and intranasally challenged with OVA either once on Day 14 or five times on Days 14 and 25 through 28. Control animals were challenged with saline. Bronchial hyperresponsiveness was measured, bronchoalveolar lavage (BAL) fluid was collected, and lungs were harvested for histology 24 h after the last challenge. The data demonstrate that wild-type (WT) mice do not respond to one OVA challenge but do develop bronchial hyperreactivity and airway and tissue eosinophilia after five OVA challenges. Conversely, Mac-1 KO mice develop significant airway eosinophilia after one OVA challenge, and the degree of airway inflammation is comparable to that observed in allergic WT mice after five challenges. In Mac-1 KO mice, after five challenges, bronchial hyperreactivity and airway inflammation was significantly enhanced compared with their wild-type counterparts. Administration of an anti-Mac-1 antibody to WT mice, before each of five intranasal OVA challenges, significantly reduces the airway eosinophilia but has no effect on tissue eosinophilia or bronchial hyperresponsiveness. Intravenous injection of interleukin-5 induced a significant blood eosinophilia in both WT and Mac-1 KO mice. Intranasal eotaxin administration induced similar levels of eosinophil migration into the lung tissues and airways of both WT and Mac-1 KO mice. In conclusion, Mac-1-deficient mice develop enhanced eosinophilic inflammation in the lung in response to allergic antigen challenge.