ISG15/USP18/STAT2 is a molecular hub regulating IFN I-mediated control of Dengue and Zika virus replication.

The establishment of a virus infection is the result of the pathogen's ability to replicate in a hostile environment generated by the host's immune system. Here, we found that ISG15 restricts Dengue and Zika viruses' replication through the stabilization of its binding partner USP18. ISG15 expression was necessary to control DV replication driven by both autocrine and paracrine type one interferon (IFN-I) signaling. Moreover, USP18 competes with NS5-mediated STAT2 degradation, a major mechanism for establishment of flavivirus infection. Strikingly, reconstitution of USP18 in ISG15-deficient cells was sufficient to restore the STAT2's stability and restrict virus growth, suggesting that the IFNAR-mediated ISG15 activity is also antiviral. Our results add a novel layer of complexity in the virus/host interaction interface and suggest that NS5 has a narrow window of opportunity to degrade STAT2, therefore suppressing host's IFN-I mediated response and promoting virus replication.

The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both In Vitro and In Vivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using in vitro and in vivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both in vitro and in vivo and reveal an opportunity for further investigation of new antituberculosis therapies.

Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

Chitosan/genipin modified electrode for voltammetric determination of interleukin-6 as a biomarker of sepsis.

Ultrasensitive electroanalytical monitoring of interleukin-6 levels in serum samples has emerged as a valuable tool for the early diagnosis of inflammatory diseases. Despite its advantages, there is a lack of strategies for the label-free voltammetric determination of cytokines. Here, a novel chitosan/genipin modified fluorine tin oxide electrode was developed providing an in-situ hydrogel formation (FTO/CSG). This platform was applied for the detection of interleukin-6, a major pro-inflammatory cytokine. Transmission electron microscopy (TEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) indicated genipin serves as an efficient green cross-linker to build the immunosensing platform (FTO/CSG/anti-IL-6). EIS showed an increase in charge transfer resistance from 326 to 1360 kΩ after the immobilization of anti-IL-6 antibodies. By square wave voltammetry, this method achieved a detection limit of 0.03 pg mL-1 with a wide linear range of 0.05-1000 pg mL-1. Additionally, it displayed a high selectivity index when tested in the presence of three inflammatory cytokines as interfering proteins: IL-12, IL-1ß, and TNF-α. The sample matrix effect showed a peak current variation lower than 5 %. The novel method was applied for the quantification of IL-6 in serum samples of septic mice. No statistical differences were observed between the standard ELISA and the proposed method using a confidence level of 95 %.

DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response.

Zika virus (ZIKV) is a single-strand RNA mosquito-borne flavivirus with significant public health impact. ZIKV infection induces double-strand DNA breaks (DSBs) in human neural progenitor cells that may contribute to severe neuronal manifestations in newborns. The DNA-PK complex plays a critical role in repairing DSBs and in the innate immune response to infection. It is unknown, however, whether DNA-PK regulates ZIKV infection. Here we investigated the role of DNA-PKcs, the catalytic subunit of DNA-PK, during ZIKV infection. We demonstrate that DNA-PKcs restricts the spread of ZIKV infection in human epithelial cells. Increased ZIKV replication and spread in DNA-PKcs deficient cells is related to a notable decrease in transcription of type I and III interferons as well as IFIT1, IFIT2, and IL6. This was shown to be independent of IRF1, IRF3, or p65, canonical transcription factors necessary for activation of both type I and III interferon promoters. The mechanism of DNA-PKcs to restrict ZIKV infection is independent of DSB. Thus, these data suggest a non-canonical role for DNA-PK during Zika virus infection, acting downstream of IFNs transcription factors for an efficient antiviral immune response.

Long-term hematopoietic stem cells as a parasite niche during treatment failure in visceral leishmaniasis.

Given the discontinuation of various first-line drugs for visceral leishmaniasis (VL), large-scale in vivo drug screening, establishment of a relapse model in rodents, immunophenotyping, and transcriptomics were combined to study persistent infections and therapeutic failure. Double bioluminescent/fluorescent Leishmania infantum and L. donovani reporter lines enabled the identification of long-term hematopoietic stem cells (LT-HSC) as a niche in the bone marrow with remarkably high parasite burdens, a feature confirmed for human hematopoietic stem cells (hHSPC). LT-HSC are more tolerant to antileishmanial drug action and serve as source of relapse. A unique transcriptional 'StemLeish' signature in these cells was defined by upregulated TNF/NF-κB and RGS1/TGF-ß/SMAD/SKIL signaling, and a downregulated oxidative burst. Cross-species analyses demonstrated significant overlap with human VL and HIV co-infected blood transcriptomes. In summary, the identification of LT-HSC as a drug- and oxidative stress-resistant niche, undergoing a conserved transcriptional reprogramming underlying Leishmania persistence and treatment failure, may open therapeutic avenues for leishmaniasis.

Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19.

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

COX-2 promotes mammary adipose tissue inflammation, local estrogen biosynthesis, and carcinogenesis in high-sugar/fat diet treated mice.

Obesity is a major risk factor for breast cancer, especially in post-menopausal women. In the breast tissue of obese women, cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production has been correlated with inflammation and local estrogen biosynthesis via aromatase. Using a mouse model of 7,12-dimethylbenz[a]anthracene/medroxyprogesterone-acetate (DMBA/MPA)-induced carcinogenesis, we demonstrated that an obesogenic diet promotes mammary tissue inflammation and local estrogen production, and accelerates mammary tumor formation in a COX-2-dependent manner. High-sugar/fat (HSF) diet augmented the levels of the pro-inflammatory mediators MCP-1, IL-6, COX-2, and PGE2 in mammary tissue, and this was accompanied by crown-like structures of breast (CLS-B) formation and aromatase/estrogen upregulation. Treatment with a COX-2 selective inhibitor, etoricoxib, decreased PGE2, IL-6, MCP-1, and CLS-B formation as well as reduced aromatase protein and estrogen levels in the mammary tissue of mice fed a HSF diet. Etoricoxib-treated mice showed increased latency and decreased incidence of mammary tumors, which resulted in prolonged animal survival when compared to HSF diet alone. Inhibition of tumor angiogenesis also seemed to account for the prolonged survival of COX-2 inhibitor-treated animals. In conclusion, obesogenic diet-induced COX-2 is sufficient to trigger inflammation, local estrogen biosynthesis, and mammary tumorigenesis.

IL-21 treatment recovers follicular helper T cells and neutralizing antibody production in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children under 1 year. RSV vaccines are currently unavailable, and children suffering from multiple reinfections by the same viral strain fail to develop protective responses. Although RSV-specific antibodies can be detected upon infection, these have limited neutralizing capacity. Follicular helper T (Tfh) cells are specialized in providing signals to B cells and help the production and affinity maturation of antibodies, mainly via interleukin (IL) 21 secretion. In this study, we evaluated whether RSV could inhibit Tfh responses. We observed that Tfh cells fail to upregulate IL-21 production upon RSV infection. In the lungs, RSV infection downregulated the expression of IL-21/interleukin-21 receptor (IL-21R) in Tfh cells and upregulated programmed death-ligand 1 (PD-L1) expression in dendritic cells (DCs) and B cells. PD-L1 blockade during infection recovered IL-21R expression in Tfh cells and increased the secretion of IL-21 in a DC-dependent manner. IL-21 treatment decreased RSV viral load and lung inflammation, inducing the formation of tertiary lymphoid organs in the lung. It also decreased regulatory follicular T cells, and increased Tfh cells, B cells, antibody avidity and neutralization capacity, leading to an overall improved anti-RSV humoral response in infected mice. Passive immunization with purified immunoglobulin G from IL-21-treated RSV-infected mice protected against RSV infection. Our results unveil a pathway by which RSV affects Tfh cells by increasing PD-L1 expression on antigen-presenting cells, highlighting the importance of an IL-21-PD-L1 axis for the generation of protective responses to RSV infection.

B1 and B2 kinin receptor blockade improves psoriasis-like disease.

BACKGROUND AND PURPOSE: The entire kallikrein-kinin system is present in the skin, and it is thought to exert a relevant role in cutaneous diseases, including psoriasis. The present study was designed to evaluate the relevance of kinin receptors in the development and progression of a model of psoriasis in mice. EXPERIMENTAL APPROACH: The effects of kinin B1 and B2 receptor knockout and of kinin receptor antagonists (SSR240612C or FR173657) were assessed in a model of psoriasis induced by imiquimod in C57BL/6 mice. Severity of psoriasis was assessed by histological and immunohistochemical assays of skin, along with objective scores based on the clinical psoriasis area and severity index. KEY RESULTS: Both kinin receptors were up-regulated following 6 days of imiquimod treatment. Kinin B1 and B2 receptor deficiency and the use of selective antagonists show morphological and histological improvement of the psoriasis hallmarks. This protective effect was associated with a decrease in undifferentiated and proliferating keratinocytes, decreased cellularity (neutrophils, macrophages, and CD4+ T lymphocytes), reduced γδ T cells, and lower accumulation of IL-17. The lack of B2 receptors resulted in reduced CD8+ T cells in the psoriatic skin. Relevantly, blocking kinin receptors reflected the improvement of psoriasis disease in the well-being behaviour of the mice. CONCLUSIONS AND IMPLICATIONS: Kinins exerted critical roles in imiquimod-induced psoriasis. Both B1 and B2 kinin receptors exacerbated the disease, influencing keratinocyte proliferation and immunopathology. Antagonists of one or even both kinin receptors might constitute a new strategy for the clinical treatment of psoriasis.

An evolutionary recent IFN/IL-6/CEBP axis is linked to monocyte expansion and tuberculosis severity in humans.

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.

Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges.

Tissue-resident macrophages are the most abundant immune cell population in healthy adipose tissue. Adipose tissue macrophages (ATMs) change during metabolic stress and are thought to contribute to metabolic syndrome. Here, we studied ATM subpopulations in steady state and in response to nutritional and infectious challenges. We found that tissue-resident macrophages from healthy epididymal white adipose tissue (eWAT) tightly associate with blood vessels, displaying very high endocytic capacity. We refer to these cells as vasculature-associated ATMs (VAMs). Chronic high-fat diet (HFD) results in the accumulation of a monocyte-derived CD11c+CD64+ double-positive (DP) macrophage eWAT population with a predominant anti-inflammatory/detoxifying gene profile, but reduced endocytic function. In contrast, fasting rapidly and reversibly leads to VAM depletion, while acute inflammatory stress induced by pathogens transiently depletes VAMs and simultaneously boosts DP macrophage accumulation. Our results indicate that ATM populations dynamically adapt to metabolic stress and inflammation, suggesting an important role for these cells in maintaining tissue homeostasis.

Vaccination with RSV M209-223 peptide promotes a protective immune response associated with reduced pulmonary inflammation.

Respiratory syncytial virus (RSV) is the most common etiologic agent in severe infections of the lower respiratory tract in children with a high mortality rate. However, there are still no licensed vaccines for RSV. In this study, we investigated a putative vaccine based on M209-223 peptide. Mice vaccinated with M209-223 peptide expanded M209-223-specific effector CD4+ T cells upon infection. Vaccination resulted in increased numbers of regulatory T cells (Treg) and Th1 cells, and decreased numbers of Th2 cells. In addition, vaccination with M209-223 peptide, protected mice from infection and prevented lung inflammation, leading to increase in IL-10 and IFN-γ production by lung CD4+ T cells. Treg depletion with anti-CTLA4 antibodies abrogated protection induced by peptide vaccination. Our results support vaccination with M209-223 peptide as an important strategy to generate protection, both systemic and local, by memory RSV-specific CD4+ T cells in mice. Contrarily to inactivated RSV particles, M209-223 peptide vaccination is capable of not only promoting viral clearance, but also reducing inflammatory processes in lungs upon infection.

The cGAS/STING Pathway Is Important for Dendritic Cell Activation but Is Not Essential to Induce Protective Immunity against Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) infection remains a major public health concern. The STING (stimulator of interferon genes) pathway contributes to the cytosolic surveillance of host cells. Most studies on the role of STING activation in Mtb infection have focused on macrophages. Moreover, a detailed investigation of the role of STING during Mtb infection in vivo is required. Here, we deciphered the involvement of STING in the activation of dendritic cells (DCs) and the host response to Mtb infection in vivo. In DCs, this adaptor molecule was important for Ifn-ß expression and IL-12 production as well as for the surface expression of the activation markers CD40 and CD86. We also documented that Mtb DNA induces STING activation in murine fibroblasts. In vivo Mtb aerogenic infection induced the upregulation of the STING and cGAS (cyclic GMP-AMP synthase) genes, and Ifn-ß pulmonary expression was dependent on both sensors. However, mice deficient for STING or cGAS presented a similar outcome to wild-type controls, with no major alterations in body weight gain, bacterial burden, or survival. Lung inflammation, proinflammatory cytokine production, and inflammatory cell recruitment were similar in STING- and cGAS-deficient mice compared to wild-type controls. In summary, although the STING pathway seems to be crucial for DC activation during Mtb infection, it is dispensable for host protection in vivo.

Human T Lymphocytes Are Permissive for Dengue Virus Replication.

Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4+ and CD8+ T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4+ and CD8+ T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4+ but not CD8+ T cells after exposure to DV in vitro Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4+ and CD8+ T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo, suggesting that this cell population may be a viral reservoir during the acute phase of the disease.IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show that both CD4+ and CD8+ T lymphocytes from acutely infected DV patients are infected by DV. Our results raise new questions about DV pathogenesis and vaccine development.

ISG15-Induced IL-10 Is a Novel Anti-Inflammatory Myeloid Axis Disrupted during Active Tuberculosis.

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.

Antibiotic-Induced Pathobiont Dissemination Accelerates Mortality in Severe Experimental Pancreatitis.

Although antibiotic-induced dysbiosis has been demonstrated to exacerbate intestinal inflammation, it has been suggested that antibiotic prophylaxis may be beneficial in certain clinical conditions such as acute pancreatitis (AP). However, whether broad-spectrum antibiotics, such as meropenem, influence the dissemination of multidrug-resistant (MDR) bacteria during severe AP has not been addressed. In the currently study, a mouse model of obstructive severe AP was employed to investigate the effects of pretreatment with meropenem on bacteria spreading and disease outcome. As expected, animals subjected to biliopancreatic duct obstruction developed severe AP. Surprisingly, pretreatment with meropenem accelerated the mortality of AP mice (survival median of 2 days) when compared to saline-pretreated AP mice (survival median of 7 days). Early mortality was associated with the translocation of MDR strains, mainly Enterococcus gallinarum into the blood stream. Induction of AP in mice with guts that were enriched with E. gallinarum recapitulated the increased mortality rate observed in the meropenem-pretreated AP mice. Furthermore, naïve mice challenged with a mouse or a clinical strain of E. gallinarum succumbed to infection through a mechanism involving toll-like receptor-2. These results confirm that broad-spectrum antibiotics may lead to indirect detrimental effects during inflammatory disease and reveal an intestinal pathobiont that is associated with the meropenem pretreatment during obstructive AP in mice.

Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1ß secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.

Isoniazid-induced control of Mycobacterium tuberculosis by primary human cells requires interleukin-1 receptor and tumor necrosis factor.

Proinflammatory cytokines are critical mediators that control Mycobacterium tuberculosis (Mtb) growth during active tuberculosis (ATB). To further inhibit bacterial proliferation in diseased individuals, drug inhibitors of cell wall synthesis such as isoniazid (INH) are employed. However, whether INH presents an indirect effect on bacterial growth by regulating host cytokines during ATB is not well known. To examine this hypothesis, we used an in vitro human granuloma system generated with primary leukocytes from healthy donors adapted to model ATB. Intense Mtb proliferation in cell cultures was associated with monocyte/macrophage activation and secretion of IL-1ß and TNF. Treatment with INH significantly reduced Mtb survival, but altered neither T-cell-mediated Mtb killing, nor production of IL-1ß and TNF. However, blockade of both IL-1R1 and TNF signaling rescued INH-induced killing, suggesting synergistic roles of these cytokines in mediating control of Mtb proliferation. Additionally, mycobacterial killing by INH was highly dependent upon drug activation by the pathogen catalase-peroxidase KatG and involved a host PI3K-dependent pathway. Finally, experiments using coinfected (KatG-mutated and H37Rv strains) cells suggested that active INH does not directly enhance host-mediated killing of Mtb. Our results thus indicate that Mtb-stimulated host IL-1 and TNF have potential roles in TB chemotherapy.