Human monocytoid cells as a model to study Toll-like receptor-mediated activation

THP-1 2A9, a subclone of the monocytoid cell line THP-1 and known to be exquisitely sensitive to LPS, was tested for TNF production following triggering by excess doses of TLR ligands. TLR2, TLR4 and TLR5 agonists, but neither TLR3 nor TLR9 agonists, induced TNF production. When used at lower concentrations, priming by calcitriol strongly influenced the sensitivity of cells to LPS and different TLR2 triggers (lipoteichoic acid (LTA), trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys) and peptidoglycan (PGN)). Priming by calcitriol failed to modulate TLR2 and TLR4 mRNA and cell surface expression of these receptors. TNF signals elicited by TLR2 agonists were blocked by the TLR-specific antibody 2392. CD14-specific antibodies showed variable effects. CD14-specific antibodies inhibited TNF induction by LTA. High concentrations partially inhibited TNF induction by Pam3Cys. The same antibodies failed to inhibit TNF induction by PGN. Thus, THP-1 2A9 cells respond by TNF production to some, but not all TLR agonists, and the wide variety of putative TLR2 agonists interact to variable degrees also with other cell-surface-expressed binding sites such as CD14. THP-1 2A9 cells might provide a model by which to investigate in more detail the interaction of pathogen-associated molecular patterns and monocytoid cell-surface-expressed pattern recognition receptors

The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacteria which infects a wide range of hosts. In ruminants, infection with L. monocytogenes frequently causes encephalitis, which is usually fatal in sheep and goat, while cattle often recover with antibiotic therapy. Since the role of NO in the control of Listeria is controversial, we have studied the expression of iNOS in the brains of cattle, sheep and goats which had succumbed to listeria encephalitis. iNOS was demonstrated in decreasing intensity in the M phi of microabscesses from cattle, sheep and goat. iNOS expression was accompanied by NT in the microabscesses of cattle, but was only present to a low degree in sheep and was absent in goats. This is indirect evidence for differences in the ability to produce NO in the three species. Presence of iNOS and NT were inversely correlated with the numbers of bacteria. While microabscesses of goats contained high amounts of L. monocytogenes they occurred only rarely in cattle. To corroborate our hypothesis that NO is involved in the control of listeria encephalitis a new animal model was developed. Eleven day old infant rats were infected intracisternally with a low dose of L. monocytogenes. This resulted in a transient meningoencephalitis with moderate clinical signs and low mortality. Listeria proliferated strongly in the inflammatory lesions during the first days of infection, reached a peak at day 4 and were eliminated until day 7. The presence of bacteria was closely accompanied by high numbers of iNOS-expressing M phi and the formation of NT. Administration of the iNOS inhibitor L-NIL or the radical scavenger PBN resulted in rapid death of the treated animals. However, the increase in bacterial numbers was one order of magnitude higher for animals treated with PBN compared with L-NIL administration. This shows that NO plays an important role in the control of a brain infection with Listeria, but suggests that reactive oxidants other than NO are also involved. In conclusion, our findings point to a possible involvement of the differences in the ability to express iNOS and subsequent NO production in the different clinical outcome of listeria encephalitis in cattle and small ruminants

Nitric oxide and metalloproteinases in canine articular ligaments: a comparison between the cranial cruciate, the medial genual collateral and the femoral head ligament

Osteoarthritis due to cranial cruciate ligament (CCL) rupture or hip dysplasia is one of the most important causes of chronic lameness in dogs. This study aimed at comparing nitric oxide (NO) production by the CCL with that of the femoral head ligament (FHL) and the medial collateral ligament (MCL), and investigating the pathway of NO production and the concomitant metalloproteinase (MMP) activity in the presence or absence of an inflammatory stimulus. Ligaments of normal dogs were subjected to different stimuli, and NO and MMP activity from explant culture supernatants were compared. The results showed that in explant cultures of the canine CCL more NO was produced than in those of the other two ligaments. A higher level of NO was produced when CCLs were exposed to the inducible nitric oxide synthase (iNOS)-inducing cocktail TNF/IL-1/LPS, and NO synthesis could be inhibited by both l-NMMA, a general nitric oxide synthase (NOS) inhibitor and l-NIL, a specific iNOS inhibitor. However, a correlation between NO synthesis and iNOS expression levels as determined by immunohistochemistry was not observed. In contrast to CCL, no evidence for iNOS-dependent NO synthesis was observed for MCL and FHL. The CCL produced less MMP than MCL and FHL, and no correlation between MMP and NO could be demonstrated. MMP activity in the CCL increased significantly after 48 h of incubation with the inflammatory stimulus. The results suggest that in canine osteoarthritis NO synthesized by canine CCL plays a more important role in the pathogenesis of osteoarthritis of the stifle than that synthesized by FHL and MCL

T Cell-Derived IL-10 Determines Leishmaniasis Disease Outcome and Is Suppressed by a Dendritic Cell Based Vaccine

Abstract In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection. Author Summary The clinical symptoms caused by infections with Leishmania parasites range from self-healing cutaneous to uncontrolled visceral disease and depend not only on the parasite species but also on the type of the host's immune response. It is estimated that 350 million people worldwide are at risk, with a global incidence of 1–1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Murine leishmaniasis is the best-characterized model to elucidate the mechanisms underlying resistance or susceptibility to Leishmania major parasites in vivo. Using T cell-specific and macrophage-specific mutant mice, we demonstrate that abrogating the secretion of the immunosuppressive cytokine IL-10 by T cells is sufficient to render otherwise susceptible mice resistant to an infection with the pathogen. The healing phenotype is accompanied by an elevated specific inflammatory immune response very early after infection. We further show that dendritic cell-based vaccination against leishmaniasis suppresses the early secretion of IL-10 following challenge infection. Thus, our study unravels a molecular mechanism critical for host immune defense, aiding in the development of an effective vaccine against leishmaniasis

Characterization of the early inflammation in <i>L. major</i>-infected T cell-specific IL-10-deficient BALB/c mice.

<p>Enhanced inflammation in T cell-specific IL-10-deficient BALB/c mice 2 weeks after infection is associated with a mixed Th1/Th2 immune response. T cell-specific IL-10 mutant and IL-10-competent BALB/c mice were infected with <i>L. major</i> promastigotes into the right hind footpad. Increase in size of the infected footpad 2 weeks after infection (<b>A</b>). Frequency of <i>L. major</i>-infected cells in the draining lymph nodes (<b>B</b>). Cytokine secretion upon restimulation of lymph node cells with <i>Leishmania</i> antigen <i>in vitro</i> (<b>C, D and E</b>). Each symbol represents an individual mouse (<b>A and B</b>); the results are representative of three independent experiments. Data show the mean ± SEM of three independent experiments with 5–6 mice per group and experiment (<b>C, D and E</b>). ** <i>p</i><0.01.</p

Characterization of the early inflammation in <i>L. major</i>-infected T cell-specific IL-10-deficient C57BL/6 mice.

<p>The early enhanced inflammation in T cell-specific and complete IL-10-deficient C57BL/6 mice is not associated with changes in the cell numbers but correlates with elevated secretion of IFN-γ by CD4⁺ and CD8⁺ cells. C57BL/6 IL-10 mutant mice were infected with <i>L. major</i> promastigotes into the right hind footpad. Increase in size of the infected compared with the non-infected footpads (<b>A</b>). Frequencies of cell populations in the infected feet (<b>B</b>). Cell numbers in the draining lymph nodes (<b>C</b>) and frequencies of cell populations in the draining lymph nodes (<b>D</b>). The results show the mean of two independent experiments with 2 mice per group and time point (<b>A to D</b>). Secretion of IFN-γ (<b>E</b>) and IL-4 (<b>F</b>) by draining lymph node cells 7 days after infection with <i>L. major</i>. Data show the mean ± SEM of four independent experiments with 2 mice per group and experiment (<b>E and F</b>). Draining lymph node cells of infected mice were stained for IFN-γ and the indicated surface markers; the fraction of double positive cells of all lymph node cells is shown. The results are representative of two experiments (<b>G</b>).</p

DC-based vaccination decreases the early secretion of IL-10 by CD4⁺ T cells following infection with <i>L. major</i>

<p>Wild-type BALB/c mice were immunized <i>in vivo</i> with fragmented BMDC that had been treated <i>in vitro</i> with <i>Leishmania</i> antigen and CpG oligodeoxynucleotides and were challenged with <i>L. major</i> promastigotes 1 week later. Control mice were mock-treated with PBS (<b>A to D</b>) or CpG activated BMDC (<b>E and F</b>). Increase in size of the infected compared with the non-infected footpad (<b>A</b>). Cytokine secretion of the draining lymph node cells upon restimulation with <i>Leishmania</i> antigen, measured by ELISA (<b>B, C and D</b>). Number of IL-10-secreting cells of all lymph node cells (<b>E</b>) and number of CD4⁺ IL-10-secreting cells of all lymph node cells (<b>F</b>) one week after infection. Data represent the mean ± SEM of three independent experiments with 2 mice per group and time point (<b>A to D</b>) or two independent experiments with 5 mice per group (<b>E and F</b>). * p<0.05.</p

Course of infection of T cell-specific, macrophage specific and complete IL-10-deficient C57BL/6 mice.

<p>T cell-specific and complete IL-10-deficient C57BL/6 mice develop enhanced inflammation despite unaltered parasite loads 7 days after infection with <i>L. major</i>. T cell-specific (CD4-Cre⁺), macrophage/neutrophil-specific (LysM-Cre⁺), complete (EIIa-Cre⁺) IL-10-deficient and IL-10-competent control mice (Cre⁻) on a C57BL/6 background were infected with <i>L. major</i> promastigotes into the right hind footpad. The increase in size of the infected compared with the non-infected footpad was measured (<b>A, B and C</b>). Mean ± SD of 8–10 mice/group (<b>A</b>). Footpad swelling 7 days after infection (<b>B</b>). Footpad swelling of T cell-specific IL-10-deficient C57BL/6 mice 7 days after injection of PBS or <i>L. major</i> parasites (<b>C</b>). Frequency of parasitized cells in the lymph nodes draining the lesions (<b>D, E and F</b>). Each symbol represents one individual mouse (<b>B, C, D and E</b>). One representative of two independent experiments is shown (<b>A, B, D and E</b>). Each time point represents the mean of two to four independent experiments with 4–6 mice in each group and experiment (<b>F</b>). * <i>p</i><0.05, ** <i>p</i><0.01. Differences in parasite load did not reach statistical significance (<b>D, E and F</b>).</p

Populations of IL-10 secreting leukocytes in lesions and draining lymph nodes early after infection with <i>L. major</i>.

<p>Following infection with <i>L. major</i>, the early secretion of IL-10 by CD4⁺ T cells in the draining lymph nodes is primarily due to FoxP3⁻/CD25⁻ T cells. Within the infected footpads, however, the low absolute number of IL-10 secreting CD4⁺ T cells is predominantly FoxP3⁺/CD25⁺. The remaining IL-10 secreting leukocytes within the infected footpads are primarily macrophages, whereas in the draining lymph nodes, also B cells and CD8⁺ T cells contribute to the overall IL-10 secretion Wild-type BALB/c mice were infected with <i>L. major</i> promastigotes into the hind footpads and 2 weeks later, cells from draining lymph nodes and lesion-derived cells were prepared. IL-10-secreting cells were identified using a cytokine secretion assay (<b>A and C</b>), and the proportion of CD4⁺ FoxP3⁺ (<b>B, D and E</b>), CD4⁺ CD25⁺ (<b>F</b>), CD8⁺, CD45⁺, F4/80⁺ or CD11c⁺ cells (<b>G</b>) was determined. The means of 4 (<b>E</b>) or 3 (<b>F and G</b>) independent experiments with 3 mice each are given.</p