Role of TLR2 and TLR4 in Human Neutrophil Functions Against Paracoccidioides brasiliensis

In paracoccidioidomycosis, a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), studies have focused on the role of neutrophils that are involved in primary response to the fungus. Neutrophil functions are regulated by pro‐ and anti‐inflammatory cytokines. The molecular mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of toll‐like receptors (TLR). We aimed at evaluating TLR2 and TLR4 expression on human neutrophils activated with GM‐CSF, IL‐15, TNF‐α or IFN‐γ and challenged with a virulent strain of P. brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H₂O₂ and IL‐6, IL‐8, TNF‐α and IL‐10 production by activated and challenged cells. All cytokines increased TLR2 and TLR4 expression. Pb18 also increased TLR2 expression inducing an additional effect to that of cytokines. On the contrary, it inhibited TLR4 expression. All cytokines increased neutrophil fungicidal activity and H₂O₂ production, but this process was not associated with TLR2 or TLR4. Neutrophils activation with GM‐CSF and TNF‐α resulted in a significative increase in IL‐8 production, while IL‐15 and IFN‐γ have no effect. Pb18 alone also increased IL‐8 production. None of the cytokines activated neutrophils for IL‐10 release. This cytokine was only detected after Pb18 challenge. Interestingly, IL‐8 and IL‐10 production involved TLR2 and mainly TLR4 modulation. Our data suggest that Pb18 uses TLR4 to gain access to human neutrophils. This interaction results in IL‐8 and IL‐10 production that may be considered as a pathogenic mechanism in paracoccidioidomycosis.     

Toll‐like receptor‐induced granulocyte‐macrophage colony‐stimulating factor secretion is impaired in Crohn's disease by nucleotide oligomerization domain 2‐dependent and ‐independent pathways

Pattern recognition receptors (PRRs) are an integral part of the innate immune system and govern the early control of foreign microorganisms. Single nucleotide polymorphisms (SNPs) in the intracellular pattern recognition receptor nucleotide‐binding oligomerization domain‐containing protein (NOD2, nucleotide oligomerization domain 2) are associated with Crohn's disease (CD). We investigated the impact of NOD2 polymorphisms on cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) in response to Toll‐like receptor (TLR) and NOD2 ligands. Based on NOD2 SNP analyses, 41 CD patients and 12 healthy controls were studied. PBMCs were stimulated with NOD2 and TLR ligands. After 18 h culture supernatants were measured using multiplex assays for the presence of human cytokines granulocyte–macrophage colony‐stimulating factor (GM‐CSF), interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α. In CD patients, TLR‐induced GM‐CSF secretion was impaired by both NOD2‐dependent and ‐independent mechanisms. Moreover, TNF‐α production was induced by a TLR‐2 ligand, but a down‐regulatory function by the NOD2 ligand, muramyl dipeptide, was impaired significantly in CD patients. Intracellular TLR ligands had minimal effect on GM‐CSF, TNF‐α and IL‐1β secretion. CD patients with NOD2 mutations were able to secrete TNF‐α, but not GM‐CSF, upon stimulation with NOD2 and TLR‐7 ligands. CD patients have impaired GM‐CSF secretion via NOD2‐dependent and ‐independent pathways and display an impaired NOD2‐dependent down‐regulation of TNF‐α secretion. The defect in GM‐CSF secretion suggests a hitherto unknown role of NOD2 in the pathogenesis of CD and is consistent with the hypothesis that impaired GM‐CSF secretion in part constitutes a NOD2‐dependent disease risk factor.     

Involvement of RP105 and Toll‐Like Receptors in the Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus

Staphylococcus aureus is the aetiological agent of many hospital‐ and community‐acquired infections. Toll‐like receptor 2 (TLR2) has been shown to play a crucial role in the host defence against S. aureus infection. The aim of this study is to investigate the roles of the heterogeneous TLR family proteins TLR2, TLR4 and RP105 during S. aureus infection. Peritoneal macrophages from mice were exposed to S. aureus. Their production of inflammatory cytokines and chemokines, their expression of cell‐surface markers and interactions between TLR2, TLR4 and RP105 were assessed in the presence or absence of inhibitory antibodies against TLR2, TLR4/MD‐2 and RP105/MD‐1 complexes. Our results demonstrate that not only TLR2 but also TLR4 and RP105 are involved in the response of macrophages to S. aureus, that TLR2, TLR4 and RP105 physically interact with each other during S. aureus infection, and that TLR2, TLR4 and RP105 both cooperate and play unique roles in the production of inflammatory cytokines (TNF‐α, IL‐12p40 and IL‐10) and chemokine (RANTES) by macrophages after S. aureus infection. This study characterizes the important roles that TLR2, TLR4 and RP105 play in host resistance against S. aureus infection.     

Activation of p38 mitogen-activated protein kinase is critical step for acquisition of effector function in cytokine-activated T cells, but acts as a negative regulator in T cells activated through the T-cell receptor

Peripheral blood CD4⁺ CD45RO⁺ T cells activated in vitro are able to induce expression of tumour necrosis factor‐α (TNF‐α) in monocytes via a contact‐dependent mechanism. Activation is achieved either with interleukin‐2 (IL‐2)/IL‐6/TNF‐α over an 8‐day period or cross‐linking CD3 using anti‐CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen‐activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti‐CD3 activated T cells, p38 MAPK is a negative regulator for anti‐CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte‐derived TNF‐α) or production of T‐cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine‐induced effector function and promotes cell proliferation and cytokine production.     

Lipoarabinomannan‐specific TNF‐α and IFN‐γ as markers of protective immunity against tuberculosis: a cohort study in an endemic setting

Lipoarabinomannan (LAM) is a virulent factor used for entry and survival of Mycobacterium tuberculosis (Mtb) in macrophages. Although the role of LAM for the diagnosis of tuberculosis (TB) has been extensively investigated, its cytokine response during natural Mtb infection in humans is largely unknown. In this study, LAM‐specific IFN‐γ, TNF‐α, and IL‐10 levels following whole blood assay were measured in untreated pulmonary TB patients, their contacts and community controls at baseline. In treated patients and contacts, cytokines were also measured at 6 and 12 months. At entry, 52.8% and 74.8% of controls and contacts were QFT‐GIT positive, respectively. At baseline, untreated TB patients and contacts had significantly lower IFN‐γ and TNF‐α response compared to community controls (p < 0.0001). Besides, untreated patients had significantly higher TNF‐α and IL‐10 response compared to their contacts (p < 0.0001). At 6 months, contacts and treated TB patients had significantly increased INF‐γ and TNF‐α response (p < 0.0001). In TB patients, IFN‐γ increased 10‐fold following chemotherapy suggesting its potential role for treatment monitoring. The data suggests that LAM might have an anti‐inflammatory effect during clinical TB and early Mtb infection. The data also suggests that LAM‐induced IFN‐γ and TNF‐α could be used as biomarkers of protective immunity.     

Antibodies Against Sporothrix schenckii Enhance TNF‐α Production and Killing by Macrophages

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell‐mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti‐gp70. Additionally, we show an increase in the levels of pro‐inflammatory cytokines such as TNF‐α and IL‐1β. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF‐α production and fungus killing by macrophages in experimental sporotrichosis.     

Mycoplasma pneumoniae lipids license TLR‐4 for activation of NLRP3 inflammasome and autophagy to evoke a proinflammatory response

Mycoplasma pneumoniae is an obligate pathogen that causes pneumonia, tracheobronchitis, pharyngitis and asthma in humans. It is well recognized that membrane lipoproteins are immunostimulants exerting as lipopolysaccharides (LPS) and play a crucial role in the pathogenesis of inflammatory responses upon M. pneumoniae infection. Here, we report that the M. pneumoniae‐derived lipids are another proinflammatory agents. Using an antibody‐neutralizing assay, RNA interference or specific inhibitors, we found that Toll‐like receptor 4 (TLR‐4) is essential for M. pneumoniae lipid‐induced tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β production. We also demonstrate that NLR family pyrin domain containing 3 inflammasome (NLRP3) inflammasome, autophagy and nuclear factor kappa B (NF‐κB)‐dependent pathways are critical for the secretion of proinflammatory cytokines, while inhibition of TLR‐4 significantly abrogates these events. Further characterization revealed that autophagy‐mediated inflammatory responses involved the activation of NF‐κB. In addition, the activation of NF‐κB promoted lipid‐induced autophagosome formation, as revealed by assays using pharmacological inhibitors, 3‐methyladenine (3‐MA) and Bay 11‐7082, or silencing of atg5 and beclin‐1. These findings suggest that, unlike the response to lipoprotein stimulation, the inflammation in response to M. pneumoniae lipids is mediated by the TLR‐4 pathway, which subsequently initiates the activation of NLRP3 inflammasome and formation of a positive feedback loop between autophagy and NF‐κB signalling cascade, ultimately promoting TNF‐α and Il‐1β production in macrophages.     

ORIGINAL ARTICLE: Effect of Progesterone on Proinflammatory Cytokine Production by Monocytes Stimulated with Pathogens Associated with Preterm birth

Problem A number of clinical trials have demonstrated that supplemental progesterone (P₄) can prevent preterm birth. Although P₄ can decrease the production of mediators of inflammation by lipopolysaccharide (LPS)‐stimulated macrophages, a majority of infections associated with preterm birth are due to Ureaplasma urealyticum, which does not contain LPS. Therefore, we studied whether P₄ could lower the production of proinflammatory cytokines by monocytes stimulated with U. urealyticum. Method of study Human monocytes (THP‐1 cells) were treated with P₄ and then stimulated with heat‐killed Escherichia coli or U. urealyticum. Cytokine concentrations in the conditioned medium were then measured by ELISA and relative viability of the cells was assessed colorimetrically. The impact of P₄ on interleukin (IL)‐1β, tumor necrosis factor‐α (TNF‐α) and IL‐8 production was assessed by comparing levels across different P₄ dosages and organism concentrations. Results Both organisms increased IL‐1β, TNF‐α and IL‐8 production in a dose‐dependent manner. P₄ enhanced the production of IL‐1β and IL‐8, but inhibited TNF‐α production by monocytes stimulated with either organism. Conclusion P₄ modulates cytokine production by E. coli and U. urealyticum‐stimulated monocytes in a similar manner and is not strictly immunosuppressive. This suggests that P₄ does not prevent preterm birth by simply suppressing bacteria‐stimulated cytokine production.     

Lipocalin‐2 ensures host defense against Salmonella Typhimurium by controlling macrophage iron homeostasis and immune response

Lipocalin‐2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron‐laden bacterial siderophores, chemo‐attracts neutrophils and has immunomodulatory and apoptosis‐regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella‐infected macrophages, which reduces cellular iron content and enhances the generation of pro‐inflammatory cytokines. Lcn2 represses IL‐10 production while augmenting Nos2, TNF‐α, and IL‐6 expression. Lcn2^?/? macrophages have elevated IL‐10 levels as a consequence of increased iron content. The crucial role of Lcn‐2/IL‐10 interactions was further demonstrated by the greater ability of Lcn2^?/? IL‐10^?/? macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2^?/? counterparts. Overexpression of the iron exporter ferroportin‐1 in Lcn2^?/? macrophages represses IL‐10 and restores TNF‐α and IL‐6 production to the levels found in wild‐type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL‐10 production, and that both functions are linked to its ability to shuttle iron from macrophages.     

Comparative analysis of spontaneous and mycobacterial antigen-induced secretion of Th1, Th2 and pro-inflammatory cytokines by peripheral blood mononuclear cells of tuberculosis patients

The cytokines produced by T helper (Th)1 cells (IFN‐γ, IL‐2 and TNF‐β) correlate with protection, whereas the cytokines released by Th2 cells (IL‐4, IL‐5) and the anti‐inflammatory cytokine IL‐10 correlate with pathogenesis of tuberculosis (TB). However, the pro‐inflammatory cytokines (IL‐1β, IL‐6, IL‐8, TNF‐α and IL‐12p70) are responsible for both protection and pathogenesis of TB. The aim of this work was to carry out a comparative analysis of cytokines present in early (day 2) and late (day 6) cultures of peripheral blood mononuclear cells (PBMCs) obtained from pulmonary tuberculosis patients. PBMCs were cultured in vitro in the absence and presence of exogenously added complex mycobacterial antigens and RD1 peptide pool. The supernatants were collected on day 2 and day 6 of culture and assayed for secreted cytokines using the flow cytomix assay. All of the cytokines, except for IL‐12p70, were spontaneously secreted by PBMCs of 27–100% TB patients, but only TNF‐α concentration was significantly higher on day 2 than day 6 (P < 0.05). Two days following antigenic stimulation, only IL‐1β, IL‐6, TNF‐α and IL‐10 were secreted in response to some mycobacterial antigens. However, 6 days later, all of the cytokines, except for IL‐2, IL‐4, IL‐5 and IL‐8, were secreted significantly in response to all complex antigens and RD1 peptides, compared with the non‐stimulated cultures (P < 0.05). In conclusion, the study shows that the longer in vitro stimulation time (6 days) was necessary for the optimal induction of IFN‐γ and TNF‐β, and practically convenient for the detection of IL‐10, IL‐1 β, TNF‐α and IL‐6.     

Nucleotide-binding oligomerization domain protein 2-deficient mice control infection with Mycobacterium tuberculosis

Nucleotide-binding oligomerization domain proteins (NODs) are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. NOD2 has recently been shown to be important for host cell cytokine responses to Mycobacterium tuberculosis, to synergize with Toll-like receptor 2 (TLR2) in mediating these responses, and thus to serve as a nonredundant recognition receptor for M. tuberculosis. Here, we demonstrate that macrophages and dendritic cells from NOD2-deficient mice were impaired in the production of proinflammatory cytokines and nitric oxide following infection with live, virulent M. tuberculosis. Mycolylarabinogalactan peptidoglycan (PGN), the cell wall core of M. tuberculosis, stimulated macrophages to release tumor necrosis factor (TNF) and interleukin-12p40 in a partially NOD2-dependent manner, and M. tuberculosis PGN required NOD2 for the optimal induction of TNF. However, NOD2-deficient mice were no more susceptible to infection with virulent M. tuberculosis than wild-type mice: they controlled the replication of M. tuberculosis in lung, spleen, and liver as well as wild-type mice, and both genotypes displayed similar lung pathologies. In addition, mice doubly deficient for NOD2 and TLR2 were similarly able to control an M. tuberculosis infection. Thus, NOD2 appears to participate in the recognition of M. tuberculosis by antigen-presenting cells in vitro yet is dispensable for the control of the pathogen during in vivo infection.     

A role for IL‐18 in protective immunity against Mycobacterium tuberculosis

Tuberculosis remains the most hazardous bacterial infection worldwide. The causative agent, Mycobacterium tuberculosis, is a facultative intracellular pathogen of resting MΦ. IFN‐γ secreted by natural killer, CD4 Th 1 and CD8 T cells upon instruction by IL‐12 and ‐18 activates MΦ to restrict mycobacterial growth. Production of both cytokines is induced by TLR signalling in DC and MΦ. Mice deficient for the TLR adaptor, MyD88, are highly susceptible to M. tuberculosis infection. Shared usage of MyD88 by signalling cascades for TLR and receptors for IL‐1 and IL‐18 prompted us to revisit the role of IL‐18 during experimental infection with M. tuberculosis. We show that mice deficient for IL‐18 and MyD88 but not for IL‐18 receptor promptly succumbed to M. tuberculosis infection in contrast to WT or TLR‐2/‐4 double KO mice indicating that lack of IL‐18 contributes to the high susceptibility of MyD88 KO mice to M. tuberculosis. Without IL‐18, the protective Th1 response was decreased and hence, mycobacterial propagation was favoured. Neutrophil‐driven lung immunopathology concomitant with unrestrained growth of tubercle bacilli are most likely responsible for the premature death of IL‐18 KO mice. Thus, IL‐18 plays a decisive role in protective immunity against tuberculosis.     

Peritoneal Cavity is a Route for Gut‐Derived Microbial Signals to Promote Autoimmunity in Non‐Obese Diabetic Mice

Macrophages play a crucial role in innate immune reactions, and peritoneal macrophages (PMs) guard the sterility of this compartment mainly against microbial threat from the gut. Type 1 diabetes (T1D) is an autoimmune disease in which gut microbiota and gut immune system appear to contribute to disease pathogenesis. We have recently reported elevated free radical production and increased permeability of gut epithelium in non‐obese diabetic (NOD) mice. Impaired barrier function could lead to bacterial leakage to the peritoneal cavity. To explore the consequences of impaired gut barrier function on extra‐intestinal immune regulation, we characterized peritoneal lavage cells from young newly weaned NOD mice. We detected a rapid increase in the number of macrophages 1–2 weeks after weaning in NOD mice compared to C57BL/6 and BALB/c mice. Interestingly, this increase in macrophages was abrogated in NOD mice that were fed an antidiabetogenic diet (ProSobee), which improves gut barrier function. Macrophages in young (5‐week‐old) NOD mice displayed a poor TNF‐α cytokine response to LPS stimulation and high expression of interleukin‐1receptor‐associated kinase‐M (IRAK‐M), indicating prior in vivo exposure to TLR‐4 ligand(s). Furthermore, injection of LPS intraperitoneally increased T cell CD69 expression in pancreatic lymph node (PaLN), suggestive of T cell activation. Leakage of bacterial components such as endotoxins into the peritoneal cavity may contribute to auto‐reactive T cell activation in the PaLN.     

Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37: effects on the production of inflammatory mediators by mouse macrophages

The ability to produce exopolysaccharides (EPS) is widespread among lactobacilli including Lactobacillus rhamnosus, the commonly used probiotic bacteria. Exopolysaccharides are a major component of the bacterial biofilm with a well‐documented impact on adherence of bacteria to host cells. However, their immunoregulatory properties are unknown. The aim of this study was to examine the immunostimulatory potential of EPS derived from L. rhamnosus KL37. We investigated the effect of EPS on the production of inflammatory mediators by mouse peritoneal macrophages and compared it with the effect of Lipopolysaccharide (LPS). Exopolysaccharides, at concentrations higher than those of LPS, stimulated production of both pro‐inflammatory (TNF‐α, IL‐6, IL‐12) and anti‐inflammatory (IL‐10) cytokines. Interestingly, analysis of the balance of TNF‐α/IL‐10 production showed a potential pro‐inflammatory effect of EPS. Furthermore, our data demonstrate that exposure of macrophages to LPS induced a state of hyporesponsiveness, as indicated by reduced production of TNF‐α after restimulation with either LPS or EPS (‘cross‐tolerance’). By contrast, EPS could make cells tolerant only to subsequent stimulation by the same stimulus. We also examined the relationship between TNF‐α production and activation of mitogen‐activated protein kinases (MAPKs) by EPS and LPS. Pretreatment of macrophages with specific inhibitors of p38 and ERK MAPKs reduced TNF‐α production induced by both stimuli to the same extent. In conclusion, these data demonstrate that EPS can effectively stimulate production of inflammatory mediators by macrophages in vitro. However, to predict whether EPS could be clinically useful as an immunomodulatory agent, further in vivo studies with highly purified EPS are necessary.     

Evidence for dispensability of protein kinase R in host control of tuberculosis

Genetic deficiency of protein kinase R (PKR) in mice was reported to enhance macrophage activation in vitro in response to interferon‐γ (IFNγ) and to reduce the burden of Mycobacterium tuberculosis (Mtb) in vivo (Wu et al. PloS One. 2012 7 :e30512). Consistent with this, treatment of wild‐type (WT) macrophages in vitro with a novel PKR inhibitor (Bryk et al., Bioorg. Med. Chem. Lett. 2011 21 :4108–4114) also enhanced IFN‐γ–dependent macrophage activation (Wu et al. PloS One. 2012 7 :e30512). Here we show that co‐treatment with IFN‐γ and a new PKR inhibitor identified herein to be highly but not completely selective likewise induced macrophages to produce more reactive nitrogen intermediates (RNI) and tumor necrosis factor alpha (TNF‐α) and less interleukin 10 (IL‐10) than seen with IFN‐γ alone. Unexpectedly, however, this new PKR inhibitor had a comparable effect on PKR‐deficient macrophages. Retrospective investigation revealed that the PKR‐deficient mice in (Wu et al. PloS One. 2012 7 :e30512) had not been backcrossed. On comparing genetically matched PKR‐deficient and WT mice, we saw no impact of PKR deficiency on macrophage activation in vitro or during the course of Mtb infection in vivo. In addition, although 129S1/SvImJ macrophage responses to IFN‐γ were greater than those of C57BL/6J macrophages, PKR was not required to mediate the IFN‐γ–dependent production of IL‐10, RNI or TNF‐α in either strain. Together the data cast doubt on PKR as a potential therapeutic target for tuberculosis.