Infliximab therapy increases the frequency of circulating CD16(+) monocytes and modifies macrophage cytokine response to bacterial infection.

Crohn's disease (CD) has been correlated with altered macrophage response to microorganisms. Considering the efficacy of infliximab treatment on CD remission, we investigated infliximab effects on circulating monocyte subsets and on macrophage cytokine response to bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, treated or not with infliximab. Macrophages were infected with Escherichia coli, Enterococcus faecalis, Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp avium, and cytokine levels [tumour necrosis factor (TNF) and interleukin (IL)-10] were evaluated at different time-points. To evaluate infliximab-dependent effects on monocyte subsets, we studied CD14 and CD16 expression by peripheral blood monocytes before and after different infliximab administrations. We also investigated TNF secretion by macrophages obtained from CD16(+) and CD16(-) monocytes and the frequency of TNF(+) cells among CD16(+) and CD16(-) monocyte-derived macrophages from CD patients. Infliximab treatment resulted in elevated TNF and IL-10 macrophage response to bacteria. An infliximab-dependent increase in the frequency of circulating CD16(+) monocytes (particularly the CD14(++) CD16(+) subset) was also observed (before infliximab: 4·65 ± 0·58%; after three administrations: 10·68 ± 2·23%). In response to MAP infection, macrophages obtained from CD16(+) monocytes were higher TNF producers and CD16(+) macrophages from infliximab-treated CD patients showed increased frequency of TNF(+) cells. In conclusion, infliximab treatment increased the TNF production of CD macrophages in response to bacteria, which seemed to depend upon enrichment of CD16(+) circulating monocytes, particularly of the CD14(++) CD16(+) subset. Infliximab treatment of CD patients also resulted in increased macrophage IL-10 production in response to bacteria, suggesting an infliximab-induced shift to M2 macrophages.

The death-promoting molecule tumour necrosis factor-related apoptosis inducing ligand (TRAIL) is not required for the development of peripheral lymphopenia or granuloma necrosis during infection with virulent Mycobacterium avium.

Disseminated infection with virulent Mycobacterium avium in C57Bl/6 (B6) mice leads to severe lymphocyte depletion in secondary lymphoid organs. In this study, we found an up-regulation of caspase-8 activity in spleen cell extracts from M. avium 25291-infected B6 mice compared to non-infected mice. The activation of this extrinsic apoptotic pathway correlated with an increase in inter-nucleosomal DNA fragmentation in CD4(+) spleen cells, as analysed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. These data suggest the involvement of death receptors in the induction of lymphocyte loss in the spleen, but previous work has excluded a role for tumour necrosis factor (TNF) receptors and Fas/CD95 in M. avium-induced lymphopenia. TNF-related apoptosis-inducing ligand (TRAIL) is expressed by different cell types of the immune system and induces apoptosis and killing of tumour cells while sparing normal cells. Here we used TRAIL(-/-) mice to determine if the absence of TRAIL prevented M. avium-induced immune pathology. We found that TRAIL-deficient mice still developed splenic lymphopenia during disseminated infection or granuloma necrosis during low-dose infections while exhibiting slightly increased susceptibility to M. avium 25291 when compared to B6 mice. However, in vivo proliferation of less virulent strains of M. avium was not influenced by TRAIL deficiency despite a decrease in interferon-γ production in infected B6.TRAIL(-/-) mice compared to B6 mice. Our results show that TRAIL does not play a significant role in either M. avium-induced pathology or protective immunity.

Constitutive expression of Bcl-2 in the haematopoietic compartment alters the metabolism of iron and increases resistance to mycobacterial infection.

Mice expressing a vav-bcl-2 transgene were tested for their resistance to an experimental infection with Mycobacterium avium. When compared with control littermates, transgenic mice exhibited an increase in the resistance to infection which was independent of B or T lymphocytes and did not require the production of gamma interferon. Macrophages from both control and transgenic mice showed equal permissiveness to M. avium growth in vitro. Finally, transgenic mice expressed diminished circulating iron levels which correlated with the increased resistance to infection.

The involvement of the chemokine receptor CXCR2 in neutrophil recruitment in LPS-induced inflammation and in Mycobacterium avium infection.

Knockout mice for CXC receptor 2 (CXCR2) chemokine receptor were used to study the recruitment of neutrophils during acute and chronic inflammatory responses. When treated with lipopolysaccharide (LPS), either intraperitoneally or intratracheally, these animals had a significant reduction in the neutrophil recruitment in the first 24-48 h as compared with control mice. During 15 days of intraperitoneal infection by Mycobacterium avium, the knockout mice showed significantly reduced numbers of neutrophils in the peritoneal cavity as compared with the control mice. In contrast, the recruitment of neutrophils to the lungs during an aerogenic M. avium infection was not affected by the CXCR2 mutation throughout the 60 days of the study. Finally, we could not find any impact of the mutation on the mycobacterial growth of the infected animals. These findings indicate that CXCR2 may be essentially involved in acute inflammatory responses where an early and rapid recruitment of neutrophils is observed.

Failure to induce enhanced protection against tuberculosis by increasing T-cell-dependent interferon-gamma generation.

We evaluated the use of recombinant human interleukin-6 (rhIL-6) and a monoclonal antibody specific for interferon-gamma (IFN-gamma) as co-adjuvants in a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis (ST-CF) emulsified in the adjuvant dimethyl-dioctadecylammonium bromide (DDA). Both the addition of rhIL-6 and the neutralization of IFN-gamma resulted in an increased T helper type 1 (Th1) response characterized by enhanced IFN-gamma production and cell proliferation. Nevertheless, this did not result in the enhancement of protection against either an intravenous or an aerosol M. tuberculosis challenge. Our data stress the need to identify further correlates of protection in addition to IFN-gamma production to screen vaccines against tuberculosis infection.

Interleukin-12 primes CD4+ T cells for interferon-gamma production and protective immunity during Mycobacterium avium infection.

Interleukin-12 (IL-12) is a crucial cytokine for the generation of a protective immune response against Mycobacterium avium infection. In contrast to infected control mice, IL-12-deficient mice were unable to control bacterial proliferation and their spleen T cells were almost unresponsive in vitro to specific antigens of M. avium. Susceptibility of mice deficient in IL-12 was similar to that of interferon-gamma (IFN-gamma)-deficient mice. These data indicate a crucial role of IL-12 in the development of a T-cell population able to produce IFN-gamma and to mediate protection against M. avium infection. Treatment of M. avium-infected mice with IL-12 induced CD4+ T cells with enhanced capacity to produce IFN-gamma as well as to confer increased protection against M. avium.

Interleukin-6 regulates the phenotype of the immune response to a tuberculosis subunit vaccine.

We investigated the role of interleukin-6 (IL-6) in the development of the immune response to a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis emulsified in the adjuvant dimethyldioctadecylammonium bromide (DDA). C57Bl/6 mice immunized with this vaccine developed a strong T helper 1 (Th1) response characterized by an increased production of interferon-gamma (IFN-gamma) secreted by CD4+ T cells. Neutralization of IL-6 during in vivo priming resulted in marked reduction in the ability of T cells to secrete IFN-gamma and IL-2 and to proliferate. IL-6 gene-disrupted mice primed with the vaccine showed a decrease in the number of IFN-gamma-producing cells and an increase in IL-4-secreting cells as compared to control mice. In contrast, neutralization of IL-6 during a boost of the vaccine in previously primed mice did not affect the development of IFN-gamma-producing cells but still increased the number of IL-4-producing cells. Our work shows that IL-6 plays a major role in the priming but not in the later expression of a Th1 response to a tuberculosis vaccine.

Blocking the receptor for IL-10 improves antimycobacterial chemotherapy and vaccination.

Novel approaches are required for the prevention and therapy of mycobacterial infections since the only vaccine in use, bacillus Calmette-Guérin, is poorly effective and chemotherapy is long and often ineffective in sterilizing the infection. We used a mouse model of Mycobacterium avium infection to address the usefulness of a mAb able to block IL-10R both in treatment of primary infections and in conventional multidrug therapy and subunit vaccination. Treatment of infected mice with this mAb during the entire period of experimental infection had little impact on the course of M. avium infection, with a slight improvement in the resistance of infected mice observed in the liver and spleen at day 30 of infection, which was associated with increased macrophage activation and priming of CD4(+) T cells for IFN-gamma production. Administration of this mAb later in infection had no effect on its course, but improved the effectiveness of chemotherapy when the latter was started in a chronic phase of infection. Also, the anti-IL-10R mAb acted as an adjuvant in the induction of protective immunity upon vaccination with a mycobacterial subunit preparation.

Effects of recombinant granulocyte-colony stimulating factor administration during Mycobacterium avium infection in mice.

Granulocyte colony-stimulating factor (G-CSF) administration in vivo has been shown to improve the defence mechanisms against infection by different microbes. Here we evaluated a possible protective role of this molecule in a mouse model of mycobacterial infection. The administration of recombinant G-CSF promoted an extensive blood neutrophilia but failed to improve the course of Mycobacterium avium infection in C57Bl/6 or beige mice. G-CSF administration also failed to improve the efficacy of a triple chemotherapeutic regimen (clarithromycin + ethambutol + rifabutin). G-CSF treatment did not protect interleukin-10 gene disrupted mice infected with M. avium. Spleen cells from infected mice treated with G-CSF had a decreased priming for antigen-specific production of interferon gamma compared to control infected mice. Our data do not substantiate previous reports on the protective activity of G-CSF in antimycobacterial immunity using mouse models.

Blocking the receptor for interleukin 10 protects mice from lethal listeriosis.

High doses of Listeria monocytogenes overcome the ability of a normal mouse to control the infection, due to massive bacterial replication. Treatment with an anti-interleukin 10 (IL-10) receptor monoclonal antibody prevented the fatal course of infection with high doses of bacteria. This work shows that blocking the receptor for IL-10 may have useful therapeutic applications.

Role of iron in experimental Mycobacterium avium infection.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) patients exhibit alterations in the metabolism of iron that lead to increased deposition of this element in the tissues. Such alterations may underlie an increased susceptibility of AIDS patients to mycobacterial infections, namely by Mycobacterium avium. OBJECTIVES: The understanding of the role of iron metabolism during M. avium infections in mouse models may allow the design of new therapies based on the manipulation of iron stores. STUDY DESIGN: In vitro macrophage cultures and in vivo mouse studies of iron depletion and iron overload are used to assess mycobacterial multiplication and testing of the efficacy of iron depletion strategies such as the use of iron chelators. RESULTS AND CONCLUSIONS: The levels of iron loading of macrophages in vitro or in vivo affect the growth of M. avium. The currently available iron chelators have poor efficacy in depleting the macrophage iron stores and, therefore, have a poor impact on the infection. Therefore, newer drugs are required that may be used in the context of in vivo infections such as in the case of affected AIDS patients.

CD8- and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis.

The role of CD8 T lymphocytes in the immune response to Mycobacterium tuberculosis infection remains enigmatic, with persuasive reports of both cytolytic and noncytolytic (cytokine-mediated) responses to infection. To address the importance of the cytolytic mechanisms, mice with targeted disruptions for CD8 and perforin or with gene mutations in the CD95/ CD95L signaling pathway were exposed to pulmonary infection. All mice tested showed no differences in their ability to contain the growth of infection during the early phase of disease. As the chronic phase of the disease ensued, however, both CD8- and CD95/CD95L-deficient mice gradually lost their ability to limit bacterial growth. This was associated with a tendency toward pyogenic inflammation in the lung. This tendency was not seen in the perforin gene-disrupted mice. In CD8 gene-disrupted mice, the ability to generate interferon-gamma secreting T cells was unimpaired. Although these cells were capable of entering the lung they were unable to influence the increasing bacterial load in this organ.

Effects of interleukin-12 in the long-term protection conferred by a Mycobacterium avium subunit vaccine.

The effects of the addition of recombinant interleukin (IL)-12 to a mycobacterial subunit vaccine were analyzed in terms of the longevity of the protective immunity generated. BALB/c mice were immunized with culture filtrate proteins from Mycobacterium avium with dimethyl-dioctadecilammonium bromide (DDA) as an adjuvant. This subunit vaccine induced protection against a challenge by M. avium which lasted for at least 6 months while waning with time until 1 year postvaccination. Whereas the addition of IL-12 enhanced the initial protective efficacy of this subunit vaccine during the first 6 months, it accelerated the loss of protective efficacy observed at 1 year postvaccination. These data confirm the adjuvant properties of IL-12 in vaccines against mycobacteria and raise the possibility of late counter-protective untoward effects.

Antigen specificity of T-cell response to Mycobacterium avium infection in mice.

T cells from Mycobacterium avium-infected C57BL/6 mice reacted to culture filtrate, envelope, and cytosol proteins and to fractions obtained from these proteins. Multiple targets were recognized, such as 29- to 45-kDa and <21-kDa antigens of the culture filtrate, antigens of around 30 kDa in the envelope and cytosol, and 45- to 116-kDa proteins in the envelope.

Modulation of neutrophil influx with cell adhesion molecule specific antibodies during nonspecific and immune mediated inflammatory reactions.

Neutrophils are essential for the host defence against infection. However, neutrophils may also mediate damage namely during immune mediated pathologies. We therefore tested whether targeting of different cell adhesion molecules with specific monoclonal antibodies might reduce immune mediated neutrophil recruitment but spare the nonspecific accumulation of neutrophils that is essential for the resistance against acute infections. Neutrophil recruitment was induced by either intraperitoneal injection of casein as a nonspecific phlogistic agent or by i.p. injection of antigen in Mycobacterium bovis Bacille Calmette-Guérin (BCG) immune mice. Similar degrees of inhibition of neutrophil accumulation were observed in both models of inflammation with antibodies directed at CD11a, ICAM-1 and CD11b with the latter showing the most marked effects. Individual targeting of selectins was without effect in immune mediated responses whereas targeting of L or E selectin inhibited nonspecific recruitment of neutrophils. This was apparently not owing to a dosage effect nor to a kinetic difference. The inhibitory effect of anti-CD11b antibodies was most likely as a result of activation of circulating neutrophils rather than the blocking of receptor-ligand interactions. We were therefore unable to selectively abrogate immune mediated neutrophil recruitment with the use of the antibodies selected in this study.

Minor role played by type I tumour necrosis factor receptor in the control of Mycobacterium avium proliferation in infected mice.

Control of mycobacterial growth depends on the concerted activity of different cytokines acting in different stages of the development of innate and adaptive immune responses. Tumour necrosis factor-alpha (TNF-alpha) has been shown to play a protective role in Mycobacterium avium infections. Here we assessed the growth of this mycobacterial species in wild-type mice and in mice with a genetically engineered disruption of the type I receptor for TNF-alpha (p55-KO mice). p55-KO mice infected with a low-virulence strain of M. avium exhibited a slightly delayed capacity to eliminate the micro-organisms from the liver as compared with wild-type animals. However, either the growth of this strain in the other organs studied (spleen and lung) or the growth of two other strains of M. avium with intermediate or high virulence, failed to be affected by mutation of the TNF-alpha receptor. p55-KO mice were also as protected by the administration of recombinant interleukin-12 as the heterozygous p55 +/- mice. We conclude that signalling through the type I TNF receptor plays a small role in vivo in the induction of mycobacteriostasis during M. avium infection but may improve survival during infection with virulent mycobacteria, independently of the extent of their proliferation.

Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice.

Evidence showing that neutrophils play a protective role in the host response to infection by different intracellular parasites has been published in the past few years. We assessed this issue with regard to the infection of mice with Mycobacterium tuberculosis. We found a chronic recruitment of neutrophils to the infection foci, namely, to the peritoneal cavity after intraperitoneal infection and to the spleen and liver after intravenous inoculation of the mycobacteria. However, bacilli were never found associated with the recruited neutrophils but rather were found inside macrophages. The intravenous administration of the antineutrophil monoclonal antibody RB6-8C5 during the first week of infection led to selective and severe neutropenia associated with an enhancement of bacillary growth in the target organs of the mice infected by the intravenous route. The neutropenia-associated exacerbation of infection was most important in the liver, where a bacterial load 10-fold higher than that in nonneutropenic mice was found; the exacerbation in the liver occurred both during and after the neutropenic period. Early in infection by M. tuberculosis, neutropenic mice expressed lower levels of mRNAs for gamma interferon and inducible nitric oxide synthase in the liver compared to nondepleted mice. These results point to a protective role of neutrophils in the host defense mechanisms against M. tuberculosis, which occurs early in the infection and is not associated with the phagocytic activity of neutrophils but may be of an immunomodulatory nature.

Mutants of Listeria monocytogenes defective in In vitro invasion and cell-to-cell spreading still invade and proliferate in hepatocytes of neutropenic mice.

Listeria monocytogenes mutants defective in the actA gene, the plcB gene, and the inlA and inlB genes were less virulent when injected intravenously into BALB/c mice. The growth of these strains as well as of the virulent wild-type strains was increased by treating mice with a neutrophil-specific depleting monoclonal antibody, RB6-8C5. Histologic examination of the livers of the treated animals showed intrahepatocytic proliferation of the listeriae in all cases. Our data show that more than one pathway exists that allows L. monocytogenes to invade parenchymal cells. One pathway most likely involves the actA and plcB gene products, and a second one probably involves the internalins.

Differences in resistance of C57BL/6 and C57BL/10 mice to infection by Mycobacterium avium are independent of gamma interferon.

After infection with a low-virulence strain of Mycobacterium avium, C57BL/6 and C57BL/10 mice had clear differences in the control of the infection in their livers and spleens. This difference in susceptibility was not associated with differences in the H-2 complex. It was dependent on the activity of CD4(+) T cells but unrelated to the ability of these cells to secrete gamma interferon or to the development of delayed-type hypersensitivity responses at 3 weeks of infection. It was associated with lower total numbers of CD4(+) cells present in infected spleens and was related to an earlier induction of protective T cells, as measured by adoptive-transfer assays. These data further strengthen the notion of gamma-interferon-independent mechanisms of protection against mycobacteria.