Despite effective antibiotic therapy, brain destructive inflammation often cannot be avoided in pneumococcal meningitis. The causative signals are mediated predominantly through TLR recruited myeloid differentiation primary response adaptor (MyD) 88 as indicated by a dramatic pneumococcal meningitis phenotype of Myd88-/- mice. Because lipoproteins and ssRNA are crucial for recognition of Gram-positive bacteria such as Streptococcus pneumoniae by the host immune system, we comparatively analyzed the disease courses of Myd88-/- and Tlr2/13-/- mice. Their phenotype resemblance indicated TLR2 and -13 as master sensors of Streptococcus pneumoniae in the cerebrospinal fluid. The neutralizing anti-TLR2 antibody (T2.5) and chloroquine (CQ) – the latter applied here as an inhibitor for murine TLR13 and its human orthologue TLR8 – abrogated activation of murine and human primary immune cells exposed to antibiotic-treated Streptococcus pneumoniae. The inhibitory effect of the T2.5/CQ cocktail was stronger than that of dexamethasone, the current standard adjunctive drug for pneumococcal meningitis. Accordingly, TLR2/13 blockade concomitant with ceftriaxone application significantly improved the clinical course of pneumococcal meningitis compared to treatment with ceftriaxone alone or in combination with dexamethasone. Our study implicates the importance of murine TLR13 and human TLR8, besides TLR2, in pneumococcal meningitis pathology, and suggests their blockade as a promising antibiotic therapy adjunct.
Susanne Dyckhoff-Shen, Ilias Masouris, Heba Islam, Sven Hammerschmidt, Barbara Angele, Veena Marathe, Jan Buer, Stefanie Völk, Hans-Walter Pfister, Matthias Klein, Uwe Koedel, Carsten J. Kirschning
With antimicrobial resistance (AMR) emerging as a major threat to global health, monoclonal antibodies (MAbs) have become a promising means to combat difficult-to-treat AMR infections. Unfortunately, in contrast with standard antimicrobials, for which there are well-validated clinical laboratory methodologies to determine whether an infecting pathogen is susceptible or resistant to a specific antimicrobial drug, no assays have been described that can inform clinical investigators or clinicians regarding the clinical efficacy of a MAb against a specific pathogenic strain. Using Acinetobacter baumannii as a model organism, we established and validated 2 facile clinical susceptibility assays, which used flow cytometry and latex bead agglutination, to determine susceptibility (predicting in vivo efficacy) or resistance (predicting in vivo failure) of 1 newly established and 3 previously described anti–A. baumannii MAbs. These simple assays exhibited impressive sensitivity, specificity, and reproducibility, with clear susceptibility breakpoints that predicted the in vivo outcomes in our preclinical model with excellent fidelity. These MAb susceptibility assays have the potential to enable and facilitate clinical development and deployment of MAbs that generally target the surface of microbes.
Matthew J. Slarve, Neven Bowler, Elizabeth Burk, Jun Yan, Ulrike Carlino-MacDonald, Thomas A. Russo, Brian M. Luna, Brad Spellberg
BACKGROUND. T cell responses are impaired in Staphylococcus aureus-infected children, highlighting a potential mechanism of immune evasion. This study tested the hypotheses that toxin-specific antibodies protect immune cells from bacterial killing and are associated with improved T cell function following infection. METHODS.S. aureus-infected and healthy children (n = 33 each) were prospectively enrolled. During acute infection and convalescence, we quantified toxin-specific IgG levels by ELISA, antibody function using a cell-killing assay, and functional T cell responses by ELISpot. RESULTS. There were no differences in toxin-specific IgG levels or ability to neutralize toxin-mediated immune cell killing between healthy and acutely-infected children, but antibody levels and function increased following infection. Similarly, T cell function, which was impaired during acute infection, improved following infection. However, the response to infection was highly variable; up to half of children did not have improved antibody or T cell function. Serum from children with higher ɑ-hemolysin (Hla)-specific IgG levels more strongly protected immune cells against toxin-mediated killing. Importantly, children whose serum more strongly protected against toxin-mediated killing also had stronger immune responses to infection, characterized by more elicited antibody and greater improvement in T cell function following infection. CONCLUSIONS. This study demonstrates that, despite T cell impairment during acute infection, S. aureus elicits toxin-neutralizing antibodies. Individual antibody responses and T cell recovery are variable. These findings also suggest that toxin-neutralizing antibodies protect antigen-presenting cells and T cells, thereby promoting immune recovery. Finally, failure to elicit toxin-neutralizing antibodies may identify children at risk for prolonged T cell suppression. FUNDING. NIAID R01AI125489 and Nationwide Children’s Hospital.
Maureen Kleinhenz, Zhaotao Li, Usha V. Chidella, Walissa Picard, Amber Wolfe, Jill Popelka, Robin Alexander, Christopher P. Montgomery
Cigarette smoking is associated with a higher risk of ICU admissions among flu patients. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice pre-exposed to CS but not room air for four weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS and flu exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest for the first time that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS induced severe flu.
Tomasz W. Kaminski, Tomasz Brzoska, Xiuying Li, Ravi Vats, Omika Katoch, Rikesh K. Dubey, Kamal Bagale, Simon C. Watkins, Bryan J. McVerry, Tirthadipa Pradhan-Sundd, Lianghui Zhang, Keven M. Robinson, Toru Nyunoya, Prithu Sundd
Tuberculosis, a chronic infectious disease caused by a single pathogen, holds the highest mortality rate worldwide. RNA-binding proteins (RBPs) are involved in autophagy — a key defense mechanism against Mycobacterium tuberculosis (Mtb) infection — by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during Mtb infection remain relatively unexplored. ZNFX1, a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in Mtb-infected macrophages. Here, we aimed to explore the immune regulatory functions of ZNFX1 during Mtb infection. We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages. This compromise resulted in reduced phagocytosis, suppressed macrophage activation, increased Mtb burden, progressive lung tissue injury, and chronic inflammation in Mtb-infected mice. Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy, consequently mediating immune suppression. ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing Prkaa2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to Mtb growth suppression. These findings reveal a function of ZNFX1 in establishing anti-Mtb immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster anti-tuberculosis immunotherapy.
Honglin Liu, Zhenyu Han, Liru Chen, Jing Zhang, Zhanqing Zhang, Yaoxin Chen, Feichang Liu, Ke Wang, Jieyu Liu, Na Sai, Xinying Zhou, Chaoying Zhou, Shengfeng Hu, Qian Wen, Li Ma
The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-β1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell–derived microglia, HSV-2–induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2–treated microglia exerted IKBKE-dependent type I IFN–mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.
Azadeh Reyahi, Marie Studahl, Morten K. Skouboe, Stefanie Fruhwürth, Ryo Narita, Fanghui Ren, Moa Bjerhem Viklund, Marie B. Iversen, Mette Christiansen, Alexandra Svensson, Trine H. Mogensen, Kristina Eriksson, Søren R. Paludan
Malaria can quickly progress from an uncomplicated infection into a life-threatening severe disease. However, the unspecificity of early symptoms often makes difficult to identify patients at high-risk of developing severe disease. Additionally, one of the most feared malaria complications -cerebral malaria- is challenging to diagnose, often resulting in treatment delays that can lead to adverse outcomes.To identify candidate biomarkers for the prognosis and/or diagnosis of severe and cerebral malaria, we have analyzed the transcriptomic response of human brain microvascular endothelial cells to erythrocytes infected with Plasmodium falciparum. Candidates were validated in plasma samples from a cohort of malaria pediatric patients from Mozambique (n = 264), resulting in the identification of several markers with capacity to distinguish uncomplicated from severe malaria, the most potent being the metallopeptidase ADAMTS18 (AUC = 0.77, P < 0.0001). Two other biomarkers, Angiopoietin-like-4 and Inhibin-βE were able to differentiate children with cerebral malaria within the severe malaria group, showing increased sensitivity after combination in a biomarker signature (AUC = 0.76, P < 0.0001).The validation of the predicted candidate biomarkers in plasma of children with severe and cerebral malaria underscores the power of this transcriptomic approach and indicates that a specific endothelial response to P. falciparum-infected erythrocytes is linked to the pathophysiology of severe malaria.
Claudia Gomes, Rosauro Varo, Miquel Duran-Frigola, Antonio Sitoe, Rubão Bila, Sonia Machevo, Alfredo Mayor, Quique Bassat, Ana Rodriguez
A better understanding of the epitopes most relevant for antibody-mediated protection against tuberculosis (TB) remains a major knowledge gap. We have shown that human polyclonal IgG to the Mycobacterium tuberculosis (Mtb) surface glycan arabinomannan (AM) and related lipoarabinomannan (LAM) is protective against TB. To investigate the impact of AM epitope recognition and Fc-gamma receptor (FcgR)-binding on antibody functions against Mtb, we isolated a high-affinity human monoclonal antibody (mAb; P1AM25) to AM and show its binding to oligosaccharide (OS) motifs we previously found to be associated with in vitro functions of human polyclonal anti-AM IgG. Human IgG1 P1AM25, but not two other high-affinity human IgG1 anti-AM mAbs reactive with different AM OS motifs, enhanced Mtb phagocytosis by macrophages and reduced intracellular growth in an FcgR-dependent manner. P1AM25 in murine IgG2a, but neither murine IgG1 nor a non-FcgR-binding IgG, given intraperitoneally prior to and after aerosolized Mtb infection was protective in C57BL/6 mice. Moreover, we demonstrate the protective efficacy of human IgG1 P1AM25 in passive transfer with Mtb-infected FcgR-humanized mice. These data enhance our knowledge of the important interplay between both antibody epitope specificity and Fc effector functions in the defense against Mtb and could inform development strategies of vaccines against TB.
Yanyan Liu, Tingting Chen, Yongqi Zhu, Aisha Furey, Todd L. Lowary, John Chan, Stylianos Bournazos, Jeffrey V. Ravetch, Jacqueline M. Achkar
Host cytosolic sensing of Mycobacterium tuberculosis (M.tb) RNA by the RIG I-like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M.tb pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M.tb pathogenesis, we evaluated M.tb intracellular growth and innate immune responses in wild-type and Mda5-/- macrophages. Transfection of M.tb RNA strongly induced pro-inflammatory cytokine production in WT macrophages, which was abrogated in Mda5-/- macrophages. M.tb infection in macrophages induced MDA5 protein expression, accompanied by an increase in MDA5 activation as assessed by multimer formation. IFNγ-primed Mda5-/- macrophages effectively contained intracellular M.tb proliferation to a significantly greater degree than WT macrophages. Further comparisons of WT versus Mda5-/- macrophages revealed that during M.tb infection MDA5 contributes to IL-1β production and inflammasome activation, and that loss of MDA5 leads to a significant increase in autophagy. In the mouse TB model, loss of MDA5 conferred host survival benefits with a concomitant reduction in M.tb bacillary burden. These data reveal that loss of MDA5 is host-protective during M.tb infection in vitro and in vivo, suggesting M.tb exploits MDA5 to subvert immune containment.
C. Korin Bullen, Alok K. Singh, Stefanie Krug, Shichun Lun, Preeti Thakur, Geetha Srikrishna, William R. Bishai
Given the resurgence of pertussis, several countries have introduced maternal tetanus, diphtheria, and acellular pertussis (aP) vaccination during pregnancy to protect young infants against severe pertussis. Although protective against the disease, the effect of maternal aP vaccination on bacterial colonization of the offspring is unknown. Here, we used a mouse model to demonstrate that maternal aP immunization, either before or during pregnancy, protects pups from lung colonization by Bordetella pertussis. However, it substantially prolongs nasal carriage by inhibiting the recruitment of IL-17-producing resident memory T cells and ensuing neutrophil influx in the nasal tissue upon B. pertussis infection, especially of those with pro-inflammatory and cytotoxic properties. Prolonged nasal carriage after aP vaccination is due to IL-4 signaling, as it is abolished in IL-4Ra-/- mice. The effect of maternal aP vaccination can be transferred transplacentally to the offspring or by breastfeeding and is long-lasting, as it persists into adulthood. Maternal aP vaccination may thus potentially augment the B. pertussis reservoir.
Violaine Dubois, Jonathan Chatagnon, Manon Depessemier, Camille Locht
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