CD209(+) macrophages mediate host defense against Propionibacterium acnes

Propionibacterium acnes is a major etiological factor of acne, triggering an inflammatory response in part through the activation of TLR2. In this study, we demonstrate that activation of peripheral blood monocytes with P. acnes in vitro induced their differentiation into two distinct innate immune cell subsets, CD209(+) macrophages and CD1b(+) dendritic cells. Furthermore, P. acnes induced expression of mRNA for the cytokines IL-15 and GM-CSF, which differentiate CD209(+) and CD1b(+) cells, respectively. The CD209(+) cells were more effective in uptake of P. acnes, compared with the CD1b(+) cells, and demonstrated a 2-fold greater antimicrobial activity against the phagocytosed bacteria. Although CD1b(+) cells secreted inflammatory cytokines in response to both P. acnes and a TLR2 ligand control, the CD209(+) cells responded only to P. acnes. The addition of all-trans retinoic acid, a commonly used agent for the treatment of acne, directly induced differentiation of monocytes into CD209(+) macrophages and enhanced the P. acnes-mediated differentiation of the CD209(+) subset. Therefore, the differentiation of monocytes into CD209(+) macrophages and CD1b(+) dendritic cells distinctly mediate the innate immune response to P. acnes.     

Enhanced resistance to Gram-positive bacterium and increased susceptibility to bacterial endotoxin in mice sensitized with Propionibacterium acnes: involvement of Toll-like receptor

Mice sensitized with Propionibacterium acnes showed an enhanced resistance against infection with Listeria monocytogenes in contrast to the increased susceptibility to LPS-induced endotoxin shock. The enhanced protection to L. monocytogenes was mediated by activated innate immunity but not by generation of Listeria-specific acquired immunity. After infection with L. monocytogenes, the elimination of bacteria was observed earlier in accordance with a higher level of endogenous cytokine production in P. acnes-sensitized mice than in control mice. Peritoneal cells from P. acnes-sensitized mice produced a larger amount of IL-12p70 and nitric oxide after stimulation with heat-killed L. monocytogenes or peptidoglycan purified from Staphylococcus aureus. RT-PCR analysis showed that the expression of TLR2 but not TLR1, TLR4 nor TLR6 was induced by injection of P. acnes in peritoneal cells. These results indicated that P. acnes-sensitization could induce the activation of innate immunity against L. monocytogenes through increased recognition of bacterial components by TLR2.     

Innate immune responses to Rhodococcus equi

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.     

Varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via Toll-like receptor 2

The pattern recognition receptor Toll-like receptor 2 (TLR2) has been implicated in the response to several human viruses, including herpes simplex viruses (types 1 and 2) and cytomegalovirus. We demonstrated that varicella-zoster virus (VZV) activates inflammatory cytokine responses via TLR2. VZV specifically induced interleukin-6 (IL-6) in human monocytes via TLR2-dependent activation of NF-kappaB, and small interfering RNA designed to suppress TLR2 mRNA reduced the IL-6 response to VZV in human monocyte-derived macrophages. Unlike other herpesviruses, the cytokine response to VZV was species specific. VZV did not induce cytokines in murine embryonic fibroblasts or in a mouse cell line, although VZV did activate NF-kappaB in a human cell line expressing a murine TLR2 construct. Together, these results suggest that TLR2 may play a role in the inflammatory response to VZV infection.     

The TLR2-MyD88-NOD2-RIPK2 signalling axis regulates a balanced pro-inflammatory and IL-10-mediated anti-inflammatory cytokine response to Gram-positive cell walls

Systemic infection with Streptococcus pneumoniae is associated with a vigorous pro-inflammatory response to structurally complex cell wall fragments (PnCW) that are shed during cell growth and antibiotic-induced autolysis. Consistent with previous studies, inflammatory cytokine production induced by PnCW was dependent on TLR2 but independent of NOD2, a cytoplasmic NLR protein. However, in parallel with the pro-inflammatory response, we found that PnCW also induced prodigious secretion of anti-inflammatory IL-10 from macrophages. This response was dependent on TLR2, but also involved NOD2 as absence of NOD2-reduced IL-10 secretion in response to cell wall and translated into diminished downstream effects on IL-10-regulated target gene expression. PnCW-mediated production of IL-10 via TLR2 required RIPK2 a kinase required for NOD2 function, and MyD88 but differed from that known for zymosan in that ERK pathway activation was not detected. As mutations in NOD2 are linked to aberrant immune responses, the temporal and quantitative effects of activation of the TLR2-NOD2-RIPK2 pathway on IL-10 secretion may affect the balance between pro- and anti-inflammatory responses to Gram-positive bacteria.     

GCN2-Dependent Metabolic Stress Is Essential for Endotoxemic Cytokine Induction and Pathology

Activated inflammatory macrophages can express indoleamine 2,3-dioxygenase (IDO) and thus actively deplete their own tryptophan supply; however, it is not clear how amino acid depletion influences macrophage behavior in inflammatory environments. In this report, we demonstrate that the stress response kinase GCN2 promotes macrophage inflammation and mortality in a mouse model of septicemia. In vitro, enzymatic amino acid consumption enhanced sensitivity of macrophages to the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) with significantly increased interleukin 6 (IL-6) production. Tryptophan withdrawal induced the stress response proteins ATF4 and CHOP/GADD153; however, LPS stimulation rapidly enhanced expression of both proteins. Moreover, LPS-driven cytokine production under amino acid-deficient conditions was dependent on GCN2, as GCN2 knockout (GCN2KO) macrophages had a significant reduction of cytokine gene expression after LPS stimulation. To test the in vivo relevance of these findings, monocytic-lineage-specific GCN2KO mice were challenged with a lethal dose of LPS intraperitoneally (i.p.). The GCN2KO mice showed reduced inflammatory responses, with decreased IL-6 and IL-12 expression correlating with significant reduction in animal mortality. Thus, the data show that amino acid depletion stress signals (via GCN2) synergize with proinflammatory signals to potently increase innate immune responsiveness.     

Toll-like receptor 4 is required for optimal development of Th2 immune responses: role of dendritic cells

LPS potently induces dendritic cell maturation and the production of proinflammatory cytokines, such as IL-12, by activation of Toll-like receptor 4 (TLR4). Since IL-12 is important for the generation and maintenance of Th1 responses and may also inhibit Th2 cell generation from naive CD4 T cell precursors, it has been inferred that TLR4 signaling would have similar effects via the induction of IL-12 secretion. Surprisingly, we found that TLR4-defective mice subjected to sensitization and pulmonary challenge with a protein allergen had reductions in airway inflammation with eosinophils, allergen-specific IgE levels, and Th2 cytokine production, compared with wild-type mice. These reduced responses were attributable, at least in part, to decreased dendritic cell function: Dendritic cells from TLR4-defective mice expressed lower levels of CD86, a costimulatory molecule important for Th2 responses. They also induced less Th2 cytokine production by antigenically naive CD4 T cells in vitro and mediated diminished CD4 T cell Ag-specific pulmonary inflammation in vivo. These results indicate that TLR4 is required for optimal Th2 responses to Ags from nonpathogenic sources and suggest a role for TLR4 ligands, such as LPS derived from commensal bacteria or endogenously derived ligands, in maturation of the innate immune system before pathogen exposure.     

Impaired recognition by Toll-like receptor 4 is responsible for exacerbated murine Pneumocystis pneumonia

We investigated the effect of Toll-like receptor 4 (TLR4) on the progression of murine Pneumocystis pneumonia. TLR4-mutant C3H/HeJ and wild-type C3H/HeN mice were infected with Pneumocystis after depletion of CD4 T cells. Mutant mice lost body weight more quickly and showed exacerbated pulmonary injury even though there was no difference in Pneumocystis organism burden in the lung. Mutant mice showed reduced levels of IL-10, IL-12p40 and MIP-2 accompanied by elevated levels of TNF-alpha and IL-6 in the bronchoalveolar lavage fluid compared with those of wild-type mice 8 weeks after the infection. In response to stimulation with Pneumocystis antigen, the production of IL-10, IL-12p40 and MIP-2 by alveolar macrophages was partially impaired in mutant mice, while that in wild-type mice was suppressed by the anti-TLR4/MD-2 mAb, MTS510. Unlike the response to lipopolysaccharide stimulation, TLR4-reconstituted HEK293 cells showed no elevated NF-kappaB activation after stimulation with Pneumocystis antigen. Taken together, these findings suggest that recognition of Pneumocystis by TLR4 helps to regulate the host inflammatory responses through cytokine and chemokine production by alveolar macrophages.     

Cutting edge: all-trans retinoic acid down-regulates TLR2 expression and function

A major consequence of microbial infection is the tissue injury that results from the host inflammatory response. In acne, inflammation is due in part to the ability of Propionibacterium acnes to activate TLR2. Because all-trans retinoic acid (ATRA) decreases inflammation in acne, we investigated whether it regulates TLR2 expression and function. Treatment of primary human monocytes with ATRA led to the down-regulation of TLR2 as well as its coreceptor CD14, but not TLR1 or TLR4. The ability of a TLR2/1 ligand to trigger monocyte cytokine release was inhibited by pre- and cotreatment with ATRA; however, TLR4 activation was affected by cotreatment only. ATRA also down-regulated monocyte cytokine induction by P. acnes. These data indicate that ATRA exerts an anti-inflammatory effect on monocytes via two pathways, one specifically affecting TLR2/1 and CD14 expression and one independent of TLR expression. Agents that target TLR expression and function represent a novel strategy to treat inflammation in humans.     

Recombinant MPT83 derived from Mycobacterium tuberculosis induces cytokine production and upregulates the function of mouse macrophages through TLR2

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.     

Polymorphisms in Toll-Like Receptor 4 Underlie Susceptibility to Tumor Induction by the Mouse Polyomavirus

PERA/Ei (PE) mice are susceptible to tumor induction by polyomavirus (Py), while C57BR/cdJ (BR) mice are resistant. Antigen-presenting cells from BR mice respond to the virus with interleukin-12 (IL-12) and those from PE mice with IL-10. These polarized cytokine responses underlie the development of effective antitumor immunity in BR mice and the lack thereof in PE mice. An ex vivo cytokine production assay using spleen cells from infected [PE × BR] F2 mice together with a genome-wide SNP (single-nucleotide polymorphism)-based QTL (quantitative trait locus) analysis was used to map the determinant of cytokine production to a region of chromosome 4 carrying the Toll-like receptor 4 (TLR4) gene. Genotyping of infected F2 mice showed concordance of TLR4 allele-specific DNA sequences with the cytokine profile. Cytokine responses elicited by Py are MyD88 dependent. Bacterial lipopolysaccharide (LPS), a known TLR4 ligand, induced the same polarized responses as the virus in these host strains. Spleen cells from C3H/HeJ and C57BL/10ScNJ LPS-nonresponsive mice challenged in vitro with Py showed an impaired IL-12 response but were unaffected in IL-10 production. TLR4s of strains PE and BR differ by 3 amino acid substitutions, 2 in the extracellular domain and 1 in the intracellular domain. cDNAs encoding the TLR4s signaled equally to an NF-κB reporter in 293 cells in a ligand-independent manner. When introduced into TLR2/TLR4 double-knockout macrophages, the TLR4 cDNA from BR mice conferred a robust IL-12 response to Py and no IL-10 response. The TLR4 cDNA from PE mice failed to confer a response with either cytokine. These results establish TLR4 as a key mediator of the cytokine response governing susceptibility to tumor induction by Py.     

Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers

The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.     

Mycobacterium abscessus activates the macrophage innate immune response via a physical and functional interaction between TLR2 and dectin-1

Mycobacterium abscessus (Mab) is an emerging and rapidly growing non-tuberculous mycobacterium (NTM). Compared with M. tuberculosis , which is responsible for tuberculosis, much less is known about NTM-induced innate immune mechanisms. Here we investigated the involvement of pattern-recognition receptors and associated signalling in Mab-mediated innate immune responses. Mab activated the extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinases (MAPKs), and induced the secretion of tumour necrosis factor-α, interleukin (IL)-6 and IL-12p40 in murine macrophages via Toll-like receptor (TLR) 2. Notably, the activation of ERK1/2, but not p38, was crucial for Mab-induced pro-inflammatory cytokine production. The ITAM-like motif of dectin-1 critically contributed to Mab internalization and cytokine secretion by macrophages. In addition, dectin-1, in cooperation with TLR2, was required for the efficient phagocytosis of Mab, ERK1/2 activation and pro-inflammatory cytokine secretion. Co-immunoprecipitation and confocal analysis showed the physical interaction and colocalization of dectin-1 with TLR2 following Mab stimulation. Moreover, dectin-1-induced Syk activation was essential for the production of inflammatory cytokines and the release of reactive oxygen species by Mab-infected macrophages. Collectively, these data demonstrate that Mab actively internalizes into and robustly activates innate immune responses in macrophages through a physical and functional interaction between TLR2 and dectin-1.     

Adjuvant effect of killed Propionibacterium acnes on mouse peritoneal B-1 lymphocytes and their early phagocyte differentiation

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

Enhanced proinflammatory response to the Candida albicans gpi7 null mutant by murine cells

The Candida albicans gpi7/gpi7 null mutant strain (Deltagpi7), which is affected in glycosylphosphatidylinositol (GPI) anchor biosynthesis, showed a reduced virulence following systemic infection of C57BL/6 mice. In vitro production of TNF-alpha, IL-6 and IL-1beta by macrophages in response to Deltagpi7 cells was significantly increased as compared to control (wild type GPI7/GPI7 and revertant gpi7/GPI7) cells; this probably contributes to the enhanced recruitment of neutrophils to the peritoneal cavity in response to Deltagpi7 cells. Survival of knockout mice for Toll-like receptor (TLR) 2 and TLR4 following intravenous injection of Deltagpi7 cells showed no significant differences as compared to C57BL/6 mice. In vitro production of TNF-alpha by macrophages and neutrophil recruitment were significantly inhibited in TLR2-/- mice in response to control yeast strains. Interestingly both TNF-alpha production and neutrophil recruitment in response to Deltagpi7 were significantly increased in all three types of mice, with no differences among them, and laminarin failed to inhibit this increased production of TNF-alpha. These results indicate that the enhanced proinflammatory response to Deltagpi7 does not involve recognition through TLR2, TLR4 nor dectin-1. Therefore, complete GPI anchors confer surface properties that are involved in modulation of cytokine production by macrophages in response to C. albicans.