Yersinia V antigen induces both TLR homo- and heterotolerance in an IL-10-involving manner

The virulence antigen (LcrV) of pathogenic yersiniae "silences" macrophages against stimulation with the TLR2-agonist zymosan A in a CD14/TLR2-dependent fashion via IL-10 induction. This pathogenically important "silencing" resembles TLR tolerance phenomena; in these, pre-exposure to a primary tolerizing TLR-agonist renders macrophages unresponsive to stimulation with a secondary challenging TLR-agonist which may involve either the same (TLR homotolerance) or a different TLR (TLR heterotolerance) as the primary TLR-agonist. Here, we show that rLcrV induces TLR homo- and heterotolerance against TLR2- or TLR4-agonists both in human and murine macrophages, respectively. The underlying mechanism of LcrV-induced tolerance is most likely not due to changes in TLR2- or TLR4 expression, but involves LcrV-mediated IL-10 production, since LcrV-induced TLR homo- and heterotolerance is highly impaired in IL-10(-/-) macrophages. Moreover, the involvement of IL-10 in TLR tolerance induction seems to be a more general phenomenon as shown by experiments using different TLR-agonists in IL-10(-/-) macrophages. Since LcrV acts as a secreted protein upon macrophages without requiring direct cell contact, as shown in transwell assays, we propose that yersiniae exploit IL-10-involving TLR tolerance mechanisms by the virulence factor LcrV.     

LcrV, a substrate for Yersinia enterocolitica type III secretion, is required for toxin targeting into the cytosol of HeLa cells

Pathogenic Yersinia species employ type III machines to transport virulence factors across the bacterial envelope. Some substrates for the type III machinery are secreted into the extracellular medium, whereas others are targeted into the cytosol of host cells. We found that during infection of tissue culture cells, yersiniae secrete small amounts of LcrV into the extracellular medium. Knockout mutations of lcrV abolish Yersinia targeting and reduce expression of the lcrGVHyopBD operon. In contrast, a block in LcrV secretion does not affect targeting, but results in premature expression and secretion of Yop proteins into the extracellular medium. LcrV-mediated activation of the type III pathway is thought to occur by sequestration of the regulatory factor LcrG, presumably via the formation of LcrV.LcrG complexes. These results suggest that intrabacterial LcrV regulates the expression and targeting of Yop proteins during Yersinia infection, whereas secreted LcrV is required to ensure specificity of Yop injection into eukaryotic cells.     

The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis

Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2-/- mice subcutaneously with Y. pestis GB. While TLR2-/- mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.     

Attachment of LcrV from Yersinia pestis at dual binding sites to human TLR-2 and human IFN-gamma receptor

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.     

LcrV is a channel size-determining component of the Yop effector translocon of Yersinia

Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact-dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127-195 of LcrV and to amino acids 106-173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size-determining structural component of the Yop translocon.     

The role of IL-10 in promoting disease progression in leishmaniasis

To determine the role of IL-10 in cutaneous leishmaniasis, we examined lesion development following Leishmania major infection of genetically susceptible BALB/c mice lacking IL-10. Whereas normal BALB/c mice developed progressive nonhealing lesions with numerous parasites within them, IL-10(-/-) BALB/c mice controlled disease progression, and had relatively small lesions with 1000-fold fewer parasites within them by the fifth week of infection. We also examined a mechanism whereby Leishmania induced the production of IL-10 from macrophages. We show that surface IgG on Leishmania amastigotes allows them to ligate Fc gamma receptors on inflammatory macrophages to preferentially induce the production of high amounts of IL-10. The IL-10 produced by infected macrophages prevented macrophage activation and diminished their production of IL-12 and TNF-alpha. In vitro survival assays confirmed the importance of IL-10 in preventing parasite killing by activated macrophages. Pretreatment of monolayers with either rIL-10 or supernatants from amastigote-infected macrophages resulted in a dramatic enhancement in parasite intracellular survival. These studies indicate that amastigotes of Leishmania use an unusual and unexpected virulence factor, host IgG. This IgG allows amastigotes to exploit the antiinflammatory effects of Fc gamma R ligation to induce the production of IL-10, which renders macrophages refractory to the activating effects of IFN-gamma.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

Toll-like receptor 6 drives differentiation of tolerogenic dendritic cells and contributes to LcrV-mediated plague pathogenesis

Educating dendritic cells (DC) to become tolerogenic DC, which promote regulatory IL-10 immune responses, represents an effective immune evasion strategy for pathogens. Yersinia pestis virulence factor LcrV is reported to induce IL-10 production via interaction with Toll-like receptor (TLR) 2. However, TLR2-/- mice are not protected against subcutaneous plague infection. Using complementary in vitro and in vivo approaches and LcrV as a model, we show that TLR6 associates with TLR2 to induce tolerogenic DC and regulatory type-1 T cells selectively secreting IL-10. In contrast, TLR1 heterodimerizes with TLR2 to promote proinflammatory IL-12p40 cytokine, producing DC and inflammatory T cell differentiation. LcrV specifically hijacks the TLR2/6 pathway to stimulate IL-10 production, which blocks host protective inflammatory responses. These results explain why TLR2 can mediate both pro- and anti-inflammatory responses and identify TLR6 as a distinct receptor driving regulatory IL-10 responses.     

Oligomerization of PcrV and LcrV, protective antigens of Pseudomonas aeruginosa and Yersinia pestis

Protective antigens of Pseudomonas aeruginosa (PcrV) and Yersinia pestis (LcrV) are key elements of specialized machinery, the type III secretion system (T3SS), which enables the injection of effector molecules into eukaryotic cells. Being positioned at the injectisome extremity, V proteins participate in the translocation process across the host cell plasma membrane. In this study, we demonstrate the assembly of V proteins into oligomeric doughnut-like complexes upon controlled refolding of the proteins in vitro. The oligomeric nature of refolded PcrV was revealed by size exclusion chromatography, native gel electrophoresis, and native mass spectrometry, which ascertain the capacity of the protein to multimerize into higher-order species. Furthermore, transmission electron microscopy performed on oligomers of both PcrV and LcrV revealed the presence of distinct structures with approximate internal and external diameters of 3-4 and 8-10 nm, respectively. The C-terminal helix, alpha12, of PcrV and notably the hydrophobic residues Val(255), Leu(262), and Leu(276) located within this helix, were shown to be crucial for oligomerization. Moreover, the corresponding mutant proteins produced in P. aeruginosa were found to be non-functional in in vivo type III-dependent cytotoxicity assays by directly affecting the correct assembly of PopB/D translocon within the host cell membranes. The detailed understanding of structure-function relationships of T3SS needle tip proteins will be of value in further developments of new vaccines and antimicrobials.     

Mutual modulation between interleukin-10 and interleukin-6 induced by Rhodococcus aurantiacus infection in mice

The interaction between interleukin-10 (IL-10) and interleukin-6 (IL-6) was investigated in the inflammatory response to Rhodococcus aurantiacus (R. aurantiacus) infection, in which both cytokines act as anti-inflammatory cytokines. Compared with wild-type (WT) counterparts, IL-6 gene-deficient (IL-6(-)/(-)) mice mounted a more robust production of IL-10 and tumor necrosis factor-alpha (TNF-alpha) during the initial phase of infection. Administration of anti-IL-10 antibody resulted in all the mice dying within 3 days post-infection as well as a further elevated TNF-alpha release. In vitro challenge of the macrophages from IL-6(-)/(-) and WT mice with heat-killed R. aurantiacus also showed similar results. Addition of exogenous IL-6 depressed IL-10 and TNF-alpha production by either IL-6(-)/(-) mice or IL-6(-)/(-) mouse macrophages. Likewise, WT mouse macrophages pretreated with anti-IL-10 or anti-IL-6 antibody exhibited increased production of TNF-alpha and IL-6 or IL-10 respectively. Moreover, neutralization of both IL-10 and IL-6 induced a further increase in TNF-alpha production by WT mouse cells. Overall, we conclude that IL-10 is a key element in protecting mice against mortality, and that IL-10 and IL-6 production are negatively regulated by each other although they are additive in suppressing TNF-alpha release in R. aurantiacus-infected mouse model.     

Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-gamma levels remained the same. TNF-alpha and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.     

Novel highly sensitive IL-10-beta-lactamase reporter mouse reveals cells of the innate immune system as a substantial source of IL-10 in vivo

In this study, we report on a novel, highly sensitive IL-10 reporter mouse based on the reporter enzyme β-lactamase and the fluorescence resonance energy transfer substrate coumarin-cephalosporin-fluorescein (4). In contrast to an IL-10 reporter mouse model that we generated by using enhanced GFP as reporter and allowed tracking IL-10 expression only in T cells, the IL-10-β-lactamase reporter (ITIB) mouse enables us to easily analyze and quantify IL-10 production at the single-cell level in all myeloid and lymphoid cell types. Furthermore, the ITIB mouse allows studying of the kinetics of IL-10 expression on a single-cell basis and provides a valuable tool for in vivo screening of cell type-specific IL-10-modulating drugs. Remarkably, the ITIB mouse revealed that, although a significant portion of each myeloid and lymphoid cell type produces IL-10, macrophages represent the major IL-10 producer population in several organs of naive mice. Moreover, using the examples of bacterial infection and transplantable skin melanoma models, we demonstrate the exceptional applicability of the ITIB mouse for the identification of IL-10-producing cells during immune responses in vivo. In this study, we identified tumor-infiltrating F4/80(+) macrophages as the major source for IL-10 in B16-F10 melanoma in vivo. During systemic infection with Yersinia enterocolitica, although the proportion of IL-10(+) cells increased in each myeloid and lymphoid cell type population, infiltrating CD11b(+)Ly6G(+) neutrophils represent a majority among IL-10-producing cells at the site of infection. We conclude that cells of the innate immune system that are involved in immune homeostasis or immune responses are substantial sources of IL-10.     

Low-dose lipopolysaccharide pretreatment suppresses choroidal neovascularization via IL-10 induction

Recent studies have suggested that some kinds of microbial infection may have a crucial role in the development of many diseases such as autoimmune diseases and certain types of cancer. It has been reported that some chronic infections, such as Chlamydia pneumoniae, and immunological dysfunctions are associated with age-related macular degeneration (AMD), a leading cause of blindness. To evaluate the association between systemic low-level inflammation induced by infection and AMD pathogenesis, we investigated whether intraperitoneal injection of lipopolysaccharide (LPS) can modulate the development of laser-induced choroidal neovascularization (CNV), a key feature of AMD. Contrary to our expectations, the sizes of CNV in mice with LPS pretreatment were approximately 65% smaller than those of the control mice. After LPS pretreatment, serum IL-10 concentration and IL-10 gene expression in peritoneal macrophages and in the posterior part of the eye increased. Peritoneal injection of anti-IL10 antibody reduced CNV suppression by LPS pretreatment. Moreover, adoptive transfer of the resident peritoneal macrophages from LPS-treated mice into control littermates resulted in an approximately 26% reduction in the size of CNV compared with PBS-treated mice. We concluded that CNV formation was suppressed by low-dose LPS pretreatment via IL-10 production by macrophages.     

Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.     

IL-10 fails to inhibit the production of IL-18 in response to inflammatory stimuli

IL-10 is an inhibitor of the production of pro-inflammatory cytokines such as IL-12 and IL-1beta, but it is not known whether it can inhibit the production of IL-18. Therefore, a variety of in vivo and in vitro models were used to determine whether IL-10 is an inhibitor of IL-18 production. Infection of IL-10-/- mice with Toxoplasma gondii results in increased levels of IL-12 in the serum and in recall responses compared to wild type (WT) mice. Surprisingly, although infection resulted in increased levels of IL-18 in serum, there were no differences between WT and IL-10-/- mice. Moreover, splenocytes from infected WT and IL-10-/- mice produced similar levels of IL-18 and addition of exogenous IL-10 did not inhibit their production of IL-18. To address whether endogenous IL-18 inhibitors were masking increased IL-18 production in the IL-10-/- mice, expression of IL-18 binding protein was examined using RT-PCR. Although infection leads to increased expression of IL-18BP mRNA, no difference was seen between WT and IL-10-/- mice. In addition, splenocytes from IL-10-/- mice produced elevated levels of nitric oxide (NO) compared to WT mice, and NO has been shown to inhibit activity of interleukin-1 converting enzyme (ICE), which is required for IL-18 production. However, the addition of an inhibitor of NO production did not alter the levels of IL-18 produced. Finally, analysis of the levels of cytokine mRNA of macrophages stimulated with LPS and IFN-gamma revealed that although IL-10 is a potent inhibitor of IL-12 mRNA accumulation, it did not inhibit IL-1beta or IL-18. Together, these data indicate that IL-10 is not an inhibitor of the production of IL-18.     

IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages

Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-alpha and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-alpha and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-alpha production, revealing for the first time a molecular mechanism underlying the parasite's suppressive effect on macrophage proinflammatory cytokine production.     

TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.