The reduction of Fusobacterium nucleatum in mice is irrelevant to the nitric oxide induced by iNOS

Previously we reported that mice infected recurrently with live Fusobacterim nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after the Fn injection. We now investigated whether the NO has the capability of killing Fn, a gram-negative rod periodontal pathogen. The mice were divided into three groups: treated with live bacteria (LB), treated with heat-killed bacteria (HKB) and untreated: normal (N). The Fn reduction, NO production and cell number after Fn injection were then compared in these mice. In the LB group, no Fn was detected at 6 hr, whereas it was still detected in the HKB and N groups at 24 hr as assessed by both colony counts and PCR assays. A significant amount of NO was synthesized in the LB group at 24 hr after the Fn injection. Fn is not killed by SNAP-generated NO in vitro. An increase in the total cell number was accompanied by an increase of the neutrophil numbers in the LB group. Intracellular O2(-) generation (including ONOO(-)) was visualized using dihydrorhodamine (DHR)-123. The peak of O2(-) generation by PEC was shown to be at 3 hr in all 3 groups. The number of O2(-) positive cells in the LB group at 3 hr was remarkably high, and most of them were likely to be neutrophils. The Fn reduction would be performed cooperatively via oxygen dependent and oxygen independent mechanisms. Thus reactive oxygen species (ROS) included in the oxygen dependent mechanism appear to be important for Fn reduction. However the significant amounts of NO derived from the iNOS synthesized in the LB group between 12 hr and 24 hr after injection of LFn were not involved in the Fn reduction.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.     

Sequential production of alpha and beta interferons and gamma interferon in the circulation of Listeria monocytogenes-infected mice after stimulation with bacterial lipopolysaccharide

Sequential production of interferon (IFN)-alpha/beta and IFN-gamma in the circulation of mice which had been previously infected with viable Listeria monocytogenes was induced by injection of lipopolysaccharide (LPS) derived from Salmonella typhimurium. IFN-alpha/beta production occurred 2 hr after injection of LPS, thereafter IFN-gamma appeared and the maximum titer was demonstrated at 6 hr. At that time, almost all of the IFN was IFN-gamma. IFN-gamma production in response to LPS was observed from the 5th through the 11th day after infection with Listeria, but it was not demonstrated in either mice infected with lower doses of viable Listeria or mice immunized with heat-killed bacteria. IFN-alpha/beta production was not drastically affected by treatment with hydrocortisone, cyclophosphamide, carrageenan, antithymocyte serum, or anti-asialo GM1 antibody, whereas IFN-gamma production was suppressed by administration of all those agents. Noteworthily, IFN-alpha/beta, but not IFN-gamma, was produced even 6 hr after stimulation with LPS in cyclophosphamide- or antithymocyte serum-treated mice. IFN-gamma induction by LPS was markedly suppressed in mice in which IFN-alpha/beta produced by Listeria infection itself had been depleted by treatment with anti-mouse IFN-alpha/beta antibody, but it was not inhibited in mice when IFN-alpha/beta induced not by Listeria infection but by LPS had been depleted by treatment with anti-mouse IFN-alpha/beta antibody.     

Differences in initial rate of intracellular killing of Salmonella typhimurium by granulocytes of Salmonella-susceptible C57BL/10 mice and Salmonella-resistant CBA mice

The contribution of granulocytes to differences in the innate susceptibility of mouse strains to infection by Salmonella typhimurium was assessed on the basis of the size and composition of the inflammatory exudate after i.p. injection of bacteria and the intracellular killing of the bacteria by exudate peritoneal cells and blood granulocytes of resistant CBA and susceptible C57BL/10 mice. The increase in the numbers of both peritoneal granulocytes and macrophages 24 hr after i.p. injection of various numbers of live S. typhimurium was two to four times higher in C57BL/10 mice (p less than 0.05) than in CBA mice. However, despite the larger number of phagocytes in the inflammatory exudate, the numbers of viable S. typhimurium in the peritoneal cavity 24 hr after injection was higher (p less than 0.01) in C57BL/10 mice than in CBA mice. Because the proportion of noningested bacteria was similar in the two mouse strains (less than 30%), these findings indicate a difference in the rate of intracellular killing of the bacteria by exudate peritoneal cells (greater than 75% granulocytes) of the two mouse strains. Subsequent determination of the initial rate of intracellular killing (Kk) of S. typhimurium revealed that after phagocytosis of the bacteria in vivo, exudate peritoneal granulocytes (harvested 24 hr after i.p. injection of 10(3) live S. typhimurium) of CBA mice killed S. typhimurium twice as efficiently (Kk = 0.014 min-1; p less than 0.01) as exudate granulocytes of C57BL/10 mice (Kk = 0.008 min-1) did. Similarly, the initial rate of intracellular killing of the ingested S. typhimurium by blood granulocytes of CBA mice (Kk = 0.017 min-1) was two times higher (p less than 0.01) than that of C57BL/10 mice (Kk = 0.007 min-1). These findings may be specific for S. typhimurium, because L. monocytogenes were killed with equal efficiency by exudate granulocytes and blood granulocytes of these mouse strains (p greater than 0.20). The results of the present study are relevant with respect to the innate resistance of mice to S. typhimurium, particularly during the initial phase of infection when the inflammatory exudate contains predominantly granulocytes.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.     

Induction of inducible nitric oxide (NO) synthase mRNA and NO production in macrophages infected with influenza A/PR/8 virus and stimulated with its ether-split product

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether-split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (M psi) cell line, and thioglycolate-elicited peritoneal M psi (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS-mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.     

Knockout of Mkp-1 enhances the host inflammatory responses to gram-positive bacteria

MAPK phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAPK. We have previously demonstrated that MKP-1 acts as a negative regulator of p38 and JNK in immortalized macrophages after stimulation with peptidoglycan isolated from Gram-positive bacteria. To define the physiological function of MKP-1 during Gram-positive bacterial infection, we studied the innate immune responses to Gram-positive bacteria using Mkp-1 knockout (KO) mice. We found that Mkp-1(-/-) macrophages exhibited prolonged activation of p38 and JNK, but not of ERK, following exposure to either peptidoglycan or lipoteichoic acid. Compared with wild-type (WT) macrophages, Mkp-1(-/-) macrophages produced more proinflammatory cytokines such as TNF-alpha and IL-6. Moreover, after challenge with peptidoglycan, lipoteichoic acid, live or heat-killed Staphylococcus aureus bacteria, Mkp-1 KO mice also mounted a more robust production of cytokines and chemokines, including TNF-alpha, IL-6, IL-10, and MIP-1alpha, than did WT mice. Accordingly, Mkp-1 KO mice also exhibited greater NO production, more robust neutrophil infiltration, and more severe organ damage than did WT mice. Surprisingly, WT and Mkp-1 KO mice exhibited no significant difference in either bacterial load or survival rates when infected with live S. aureus. However, in response to challenge with heat-killed S. aureus, Mkp-1 KO mice exhibited a substantially higher mortality rate compared with WT mice. Our studies indicate that MKP-1 plays a critical role in the inflammatory response to Gram-positive bacterial infection. MKP-1 serves to limit the inflammatory reaction by inactivating JNK and p38, thus preventing multiorgan failure caused by exaggerated inflammatory responses.     

The significance of alpha/beta interferons and gamma interferon produced in mice infected with Listeria monocytogenes

Interferon (IFN)-alpha/beta was induced in the circulation of mice infected intravenously with Listeria monocytogenes 24 to 72 hr after infection, but was not induced by the administration of heat-killed Listeria, listerial cell wall fraction (LCWF), or listerial soluble fraction. Appearance of IFN-alpha/beta showed a pattern similar to that of the growth of bacteria in the spleen and the liver of mice. IFN-alpha/beta production was abrogated by pretreatment of mice with anti-asialo GM1 antibody, antithymocyte serum, or hydrocortisone, but not with cyclophosphamide or carrageenan. Such treatments which suppressed IFN-alpha/beta production did not influence bacterial growth in the organs of mice in the early stage of Listeria infection. Administration of IFN-alpha/beta exogenously also did not. After 5 days of infection when the specific resistance against reinfection with Listeria was established, IFN-gamma but not IFN-alpha/beta was induced in the circulation 3 to 6 hr after stimulation with LCWF or reinfection with Listeria. IFN-gamma production was abrogated completely by cyclophosphamide and antithymocyte serum, and partially by hydrocortisone and carrageenan, but not by anti-asialo GM1 antibody in Listeria-infected mice treated with these agents before induction of IFN-gamma by LCWF. Presumably, IFN-alpha/beta might be produced by asialo GM1-bearing cells but IFN-gamma might not. However, IFN-gamma production was suppressed in Listeria-infected mice, when IFN-alpha/beta production had been inhibited by treatment with anti-asialo GM1 antibody or when the IFN produced had been neutralized with anti-mouse IFN-alpha/beta antibody. Therefore, it is conceivable that IFN-alpha/beta might be essential for the generation or the expression of antigen-specific T cells involving IFN-gamma production and acquired resistance during Listeria infection. In fact, the bacterial growth in the organs of mice in the early stage of infection was normal in IFN-alpha/beta-depleted mice but it resulted in the delay of T-cell-dependent elimination of bacteria from the organs of mice in the late stage.     

IL-17A synergizes with IFN-γ to upregulate iNOS and NO production and inhibit chlamydial growth

IFN-γ-mediated inducible nitric oxide synthase (iNOS) expression is critical for controlling chlamydial infection through microbicidal nitric oxide (NO) production. Interleukin-17A (IL-17A), as a new proinflammatory cytokine, has been shown to play a protective role in host defense against Chlamydia muridarum (Cm) infection. To define the related mechanism, we investigated, in the present study, the effect of IL-17A on IFN-γ induced iNOS expression and NO production during Cm infection in vitro and in vivo. Our data showed that IL-17A significantly enhanced IFN-γ-induced iNOS expression and NO production and inhibited Cm growth in Cm-infected murine lung epithelial (TC-1) cells. The synergistic effect of IL-17A and IFN-γ on Chlamydia clearance from TC-1 cells correlated with iNOS induction. Since one of the main antimicrobial mechanisms of activated macrophages is the release of NO, we also examined the inhibitory effect of IL-17A and IFN-γ on Cm growth in peritoneal macrophages. IL-17A (10 ng/ml) synergizes with IFN-γ (200 U/ml) in macrophages to inhibit Cm growth. This effect was largely reversed by aminoguanidine (AG), an iNOS inhibitor. Finally, neutralization of IL-17A in Cm infected mice resulted in reduced iNOS expression in the lung and higher Cm growth. Taken together, the results indicate that IL-17A and IFN-γ play a synergistic role in inhibiting chlamydial lung infection, at least partially through enhancing iNOS expression and NO production in epithelial cells and macrophages.     

Activation of the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase by macrophage-stimulating protein results in the induction of arginase activity in murine peritoneal macrophages.

Regulation of macrophage activities in response to inflammatory stimuli must be finely tuned to promote an effective immune response while, at the same time, preventing damage to the host. Our lab and others have previously shown that macrophage-stimulating protein (MSP), through activation of its receptor RON, negatively regulates NO production in response to IFN-gamma and LPS by inhibiting the expression of inducible NO synthase (iNOS). Furthermore, activated macrophages from mice harboring targeted mutations in RON produce increased levels of NO both in vitro and in vivo, rendering them more susceptible to LPS-induced endotoxic shock. In this study, we demonstrate that stimulation of murine peritoneal macrophages with MSP results in the RON-dependent up-regulation of arginase, an enzyme associated with alternative activation that competes with iNOS for the substrate L-arginine, the products of which are involved in cell proliferation and matrix synthesis. Expression of other genes associated with alternative activation, including scavenger receptor A and IL-1R antagonist, is also up-regulated in MSP-stimulated murine macrophages. Stimulation of cells with IFN-gamma and LPS blocks the ability of MSP to induce arginase activity. However, pretreatment of cells with MSP results in the up-regulation of arginase and inhibits their ability to produce NO in response to IFN-gamma and LPS, even in the presence of excess substrate, suggesting that the inhibition of NO by MSP occurs primarily through its ability to regulate iNOS expression.     

Kinase suppressor of Ras-1 protects against pulmonary Pseudomonas aeruginosa infections

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe infections in immunocompromised individuals and individuals with cystic fibrosis or chronic obstructive pulmonary disease (COPD). Here we show that kinase suppressor of Ras-1 (Ksr1)-deficient mice are highly susceptible to pulmonary P. aeruginosa infection accompanied by uncontrolled pulmonary cytokine release, sepsis and death, whereas wild-type mice clear the infection. Ksr1 recruits and assembles inducible nitric oxide (NO) synthase (iNOS) and heat shock protein-90 (Hsp90) to enhance iNOS activity and to release NO upon infection. Ksr1 deficiency prevents lung alveolar macrophages and neutrophils from activating iNOS, producing NO and killing bacteria. Restoring NO production restores the bactericidal capability of Ksr1-deficient lung alveolar macrophages and neutrophils and rescues Ksr1-deficient mice from P. aeruginosa infection. Our findings suggest that Ksr1 functions as a previously unknown scaffold that enhances iNOS activity and is therefore crucial for the pulmonary response to P. aeruginosa infections.     

Type I IFNs stimulate nitric oxide production and resistance to Trypanosoma cruzi infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-alphabeta or anti-TNF-alpha Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-alpha (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-alphabeta Ab reduced NO production. IFN-gamma or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-alphabetaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-alphabeta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.     

IFN-gamma-dependent nitric oxide production is not linked to resistance in experimental African trypanosomiasis

Resistance to African trypanosomes is dependent on B cell and Th1 cell responses to the variant surface glycoprotein (VSG). While B cell responses to VSG control levels of parasitemia, the cytokine responses of Th1 cells to VSG appear to be linked to the control of parasites in extravascular tissues. We have recently shown that IFN-gamma knockout (IFN-gamma KO) mice are highly susceptible to infection and have reduced levels of macrophage activation compared to the wild-type C57BL/6 (WT) parent strain, even though parasitemias were controlled by VSG-specific antibody responses in both strains. In the present work, we examine the role of IFN-gamma in the induction of nitric oxide (NO) production and host resistance and in the development of suppressor macrophage activity in mice infected with Trypanosoma brucei rhodesiense. In contrast to WT mice, susceptible IFN-gamma KO mice did not produce NO during infection and did not develop suppressor macrophage activity, suggesting that NO might be linked to resistance but that suppressor cell activity was not associated with resistance or susceptibility to trypanosome infection. To further examine the consequence of inducible NO production in infection, we monitored survival, parasitemia, and Th cell cytokine production in iNOS KO mice. While survival times and parasitemia of iNOS KO mice did not differ significantly from WT mice, VSG-specific Th1 cells from iNOS KO mice produced higher levels of IFN-gamma and IL-2 than cells from WT mice. Together, these results show for the first time that inducible NO production is not the central defect associated with susceptibility of IFN-gamma KO mice to African trypanosomes, that IFNgamma-induced factors other than iNOS may be important for resistance to the trypanosomes, and that suppressor macrophage activity is not linked to either the resistance or the susceptibility phenotypes.     

Macrophages in resistance to rickettsial infection: macrophage activation in vitro for killing of Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi, strain Gilliam, replicates in cultures of resident peritoneal macrophages from BALB/c mice. Macrophage cultures treated with culture supernatants of spleen cells from rickettsial-infected mice stimulated with heat-killed rickettsiae markedly suppressed macrophage infection by rickettsiae. Rickettsiacidal activity of activated macrophages was dependent upon both lymphokine concentration and time of incubation in lymphokines. Treatment of macrophage cultures with lymphokines before exposure to viable rickettsiae resulted in an immediate decrease in percent macrophages infected and numbers of viable intracellular rickettsiae. In these cultures, enhanced intracellular killing was also apparent with further incubation (24 hr). The immediate effect of lymphokine-pretreated macrophages was dissociated from intracellular killing by infecting macrophage cultures first and adding lymphokines after infection. In these cultures, both percent macrophages infected and titers of viable intracellular rickettsiae were dramatically reduced as well.     

Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFbeta-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-gamma and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-beta1 and IL-4 production by BALB/c mice and to an increase in the IFN-gamma levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.     

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.     

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3-5 days after infection, and is undetectable by 7-10 days. During the period of HSV-1 replication, macrophages and gammadelta TCR+ T lymphocytes infiltrate the TG, and TNF-alpha, IFN-gamma, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-alpha, IFN-gamma, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and gammadelta TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-alpha and iNOS, whereas gammadelta TCR+ T cells produce IFN-gamma. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-alpha or IFN-gamma all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-alpha or IFN-gamma markedly reduced the accumulation of both macrophages and gammadelta TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and gammadelta TCR+ T lymphocytes through their production of antiviral molecules TNF-alpha, NO, and IFN-gamma. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.     

Absence of involvement of nitric oxide in LP-BM5-induced immunodeficiency syndrome

Abstract To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of l -NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFNγ + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by l -NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.