Bacterial Burden Research Articles

Unraveling host-pathogen dynamics in a murine model of septic peritonitis induced by vancomycin-resistant Enterococcus faecium

ABSTRACT Vancomycin-resistant Enterococcus faecium (E. faecium) infection is associated with higher mortality rates. Previous studies have emphasized the importance of innate immune cells and signalling pathways in clearing E. faecium, but a comprehensive analysis of host-pathogen interactions is lacking. Here, we investigated the interplay of host and E. faecium in a murine model of septic peritonitis. Following injection with a sublethal dose, we observed significantly increased murine sepsis score and histological score, decreased weight and bacterial burden, neutrophils and macrophages infiltration, and comprehensive activation of cytokine-mediated signalling pathway. In mice receiving a lethal dose, hypothermia significantly improved survival, reduced bacterial burden, cytokines, and CD86 expression of MHC-II+ recruited macrophages compared to the normothermia group. A mathematical model constructed by observational data from 80 animals, recapitulated the host-pathogen interplay, and further verified the benefits of hypothermia. These findings indicate that E. faecium triggers a severe activation of cytokine-mediated signalling pathway, and hypothermia can improve outcomes by reducing bacterial burden and inflammation.

RANKL-mediated osteoclast formation is required for bone loss in a murine model of Staphylococcus aureus osteomyelitis

Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition.

Integration of individual preclinical and clinical anti-infective PKPD data to predict clinical study outcomes.

The AIDA randomized clinical trial found no significant difference in clinical failure or survival between colistin monotherapy and colistin-meropenem combination therapy in carbapenem-resistant Gram-negative infections. The aim of this reverse translational study was to integrate all individual preclinical and clinical pharmacokinetic-pharmacodynamic (PKPD) data from the AIDA trial in a pharmacometric framework to explore whether individualized predictions of bacterial burden were associated with the trial outcomes. The compiled dataset included for each of the 207 patients was (i) information on the infecting Acinetobacter baumannii isolate (minimum inhibitory concentration, checkerboard assay data, and fitness in a murine model), (ii) colistin plasma concentrations and colistin and meropenem dosing history, and (iii) disease scores and demographics. The individual information was integrated into PKPD models, and the predicted change in bacterial count at 24 h for each patient, as well as patient characteristics, was correlated with clinical outcomes using logistic regression. The invivo fitness was the most important factor for change in bacterial count. A model-predicted growth at 24 h of ≥2-log10 (164/207) correlated positively with clinical failure (adjusted odds ratio, aOR = 2.01). The aOR for one unit increase of other significant predictors were 1.24 for SOFA score, 1.19 for Charlson comorbidity index, and 1.01 for age. This study exemplifies how preclinical and clinical anti-infective PKPD data can be integrated through pharmacodynamic modeling and identify patient- and pathogen-specific factors related to clinical outcomes - an approach that may improve understanding of study outcomes.

Slaughtering processes impact microbial communities and antimicrobial resistance genes of pig carcasses

Several steps in the abattoir can influence the presence of microbes and associated resistance genes (ARGs) on the animal carcasses used for further meat processing. We investigated how these processes influence the resistome-microbiome of groups of pigs with different on-farm antimicrobial exposure status, from the moment they entered the abattoir until the end of carcass processing. Using a targeted enrichment metagenomic approach, we identified 672 unique ARGs conferring resistance to 43 distinct AMR classes from pooled skin (N = 42) and carcass swabs (N = 63) collected sequentially before, during, and after the slaughter process and food safety interventions. We observed significant variations in the resistome and microbial profiles of pigs before and after slaughter, as well as a significant decline in ARG counts, diversity, and microbial DNA load during slaughter and carcass processing, irrespective of prior antimicrobial treatments on the farm. These results suggest that existing interventions in the abattoir are effective in reducing not only the pathogen load but also the overall bacterial burden, including ARGs on pork carcasses. Concomitant with reductions in microbial and ARG counts, we observed an increase in the relative abundance of non-drug-specific ARGs, such as those conferring resistance to metals and biocides, and in particular mercury. Using a strict colocalization procedure, we found that most mercury ARGs were associated with genomes from the Pseudomonadaceae and Enterobacteriaceae families. Collectively, these findings demonstrate that slaughter and processing practices within the abattoir can shape the microbial and ARG profiles of pork carcasses during the transition from living muscle to meat.

Tristetraprolin mediates immune evasion of mycobacterial infection in macrophages.

Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA-binding proteins controlling the stability of targeted mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in intracellular mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by the mTOR inhibitor rapamycin. Rapamycin and AZD8055 specifically blocked 4EBP1 phosphorylation in infected macrophages and suppressed intracellular BCG growth. Rapamycin promoted TTP protein degradation through the ubiquitination pathway, whereas the proteasome inhibitor MG-132 blocked rapamycin function and thus stabilized TTP protein. TTP induction suppressed the expression of iNOS/TNF-α/IL-12/IL-23, and weakened protective immune responses in macrophages, whereas rapamycin enhanced the bactericidal effects through TTP inhibition. Moreover, blocking TTP binding increased the expression of TNF-α and iNOS and suppressed intracellular mycobacterial growth. Overall, our study reveals a novel role for RNA-binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host-directed therapy against tuberculosis (TB).

Substance P receptor signaling contributes to host maladaptive responses during enteric bacterial infection.

Immune responses in the intestine are intricately balanced to prevent pathogen entry without inducing immunopathology. The nervous system is well-established to interface with the immune system to fine-tune immunity in various organ systems including the gastrointestinal tract. Specialized sensory neurons can detect bacteria, bacterial products, and the resulting inflammation, to coordinate the immune response in the gastrointestinal tract. These sensory neurons release peptide neurotransmitters such as Substance P (SP), to induce both neuronal signaling and localized responses in non-neuronal cells. With this in mind, we assessed the immunoregulatory roles of SP receptor signaling during enteric bacterial infection with the non-invasive pathogen Citrobacter rodentium. Pharmacological antagonism of the SP receptor significantly reduced bacterial burden and prevented colonic crypt hyperplasia. Mice with SP receptor signaling blockade had significantly reduced inflammation and recruitment of T-cells in the colon. Reduced colonic T-cell recruitment is due to reduced expression of adhesion molecules on colonic endothelial cells in SP receptor antagonist-treated mice. Using SP receptor T-cell conditional knockout mice, we further confirmed SP receptor signaling enhanced select aspects of T-cell responses. Our data demonstrates that SP receptor signaling can significantly reduce inflammation and prevent host-maladaptive responses without impinging upon host protection.

The disruptive effect on the bacterial cell wall explored of the ultrasmall particle-sized Ag nanoparticles supported with Zn Cu-based hybrid nanoagents

The smaller the particle size of Ag nanoparticles, the higher their antibacterial activity against drug-resistant strains, and the greater their potential for application in living organisms. Therefore, preparing Ag nanoparticles with ultra-small particle sizes has become one of the greatest challenges at present. In this study, 1.75 nm Ag was uniformly anchored on the surface of a highly biocompatible carrier to synthesize Fe3O4/ZnO@Cu2S@Ag (FCA) nanocomposites. The antibacterial activity and mechanism of FCA were investigated, and the material was used to treat wound infections caused by bacteria. At a concentration of 175 μg/mL, the antibacterial rate of Escherichia coli, Staphylococcus aureus, and drug-resistant Salmonella was over 99.99 % within 45 min. The antibacterial activity is mainly composed of •OH and multiple cations. The antibacterial mechanism involves •OH and multiple cations synergistically targeting and damaging the bacterial cell wall, altering the secondary structure of the bacterial cell wall, leading to leakage of bacterial contents and bacterial death. Additionally, FCA exhibited high biocompatibility, curing bacterial infections within 7 days and completely eliminating bacterial burden. These findings elucidate the role of antibacterial activity in composite antibacterial materials and suggest that FCA has clinical application potential in treating bacterial infections.

Baicalein loaded liposome with hyaluronic acid and Polyhexamethylene guanidine modification for anti methicillin-resistant Staphylococcus aureus infection

Targeting delivery to the infection site and good affinity of vehicle to the bacterial are two main concerns in therapy of bacterial infection, and on-demand release of drug is another important issue. In this work, a liposome drug delivery system (HA/P/BAI-lip) incorporated with baicalein and modified by PHMG and HA was prepared. Several characterizations were conducted to examine the physical properties of liposome. Then it was applied to treatments of MRSA induced dorsal subcutaneous abscess model and the thigh muscle infected model. The presence of guanidine group in HA/P/BAI-lip rendered the liposome satisfactory bacterial target ability and good pH sensitive properties. The lipase secreted by bacterial could promote the hydrolysis of soybean phosphatidylcholine (SPC) in liposome. The modification of HA in HA/P/BAI-lip could lead the drug system to the exact infected site where CD44 was abundant because of inflammation. The low pH microenvironment characteristic of bacterial infection could induce the swelling of liposome following by degradation. Taken together, baicalein could be released selectively at the infected site to exert antibacterial capacity. HA/P/BAI-lip showed impressive antibacterial ability and dramatically decrease the bacterial burden of infection site and alleviate the infiltration of inflammatory cells, facilitating the recovery of infection.

Rapid bacterial evaluation beyond the colony forming unit in osteomyelitis.

Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony forming unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.

Research Note: Synergistic effect of isopropoxy benzene guanidine and colistin against mcr-1-positive Escherichia coli in vitro and in duck intestine infection models

Colistin (CST) is considered as "agent of last resort" against gram-negative bacteria as feed additive. Its clinical effectiveness has reduced since the emergence of mcr-1 gene in ducks. Isopropoxy benzene guanidine (IBG), a new guanidine derivative, showed positive effects on improving animal weights and alleviating intestinal pathogens, therefore, the objective of this study was to evaluate the effect of this compound supplement with CST in ducks and explore the possibilities in feed additive. A total of fifteen duck-origin Escherichia coli carrying the mcr-1 gene were included in this study. A checkerboard microdilution assay was used to evaluate the in vitro antibacterial activity of IBG combined with CST against mcr-1-positive E. coli. A 3-by-2 time-kill array of IBG (16, 32, and 64 μg/mL) and CST (1/2 MIC and 1/4 MIC) over 24 hours was utilized to characterize the activity of the agents alone and in combination against E. coli strain 1 in vitro. The intestinal colonization model was used to evaluate the in vivo effect of IBG combined with CST. These results indicated that the combination of IBG plus CST showed a synergistic effect against all clinical isolates (FICI < 0.5). The bacterial burden was reduced by more than 2 log10 CFU/mL when E. coli strain 1 was tested with 1/2 MIC CST plus 64 μg/mL IBG for 24 h. Further experiments in vivo demonstrated that the CST combined with IBG was able to increase duck weights, reduced intestinal pathogenic E. coli and showed a synergistic antibacterial effect. Combination of CST (4 mg/kg b.w.) plus IBG (32 or 64 mg/kg b.w.) achieved 1.84 to 3.29 log10 CFU/g killing after 7 d of therapy, which was significantly different from that in the challenge control group (p<0.05). In summary, our study demonstrated the potential use of IBG as feed additive for veterinary purposes in ducks and provided new insights into overcoming resistance in the future.

Rapid bacterial evaluation beyond the colony forming unit in osteomyelitis

Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony forming unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.

Long-term Outcomes of Adjunctive Lung Resection for Nontuberculous Mycobacteria Pulmonary Disease.

Adjunctive lung resection is recommended for select patients with nontuberculous mycobacteria (NTM) pulmonary disease (PD). However, data are limited on long-term recurrence rates in patients infected with major pathogens, including Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MABC). In this prospective observational study, we retrospectively analyzed data from 125 patients with MAC-PD (n = 90) or MABC-PD (n = 35) who underwent adjunctive lung resection. We evaluated microbiological response, postoperative complications, recurrence, and all-cause mortality over a median 80-month follow-up. Persistent culture positivity (64%) was the most common indication for surgery, followed by hemoptysis, recurrent pneumonia, or radiologic deterioration. Postoperative complications occurred in 18 (14%) patients, with no surgery-related deaths. Treatment outcomes did not significantly differ between the MAC- and MABC-PD groups. Cure with culture conversion was achieved in 112 (90%) patients. Recurrence occurred in 37 (33%) of 112 patients, of which 18 (49%) cases were attributed to reinfection by different NTM species or subspecies. The MAC group had higher recurrence rates than the MABC group (Kaplan-Meier curve, log-rank test, P = .043) and was significantly associated with recurrence in the multivariable analysis (adjusted hazard ratio, 2.71; 95% CI, 1.23-5.99). However, mortality was higher in the MABC-PD group than the MAC-PD group (7/35 vs 4/90, P = .006). Adjunctive lung resection with antibiotics helps to reduce bacterial burden and manage symptoms in patients with NTM-PD. However, it does not prevent recurrence, which is mostly caused by reinfection.

CD5L is upregulated upon infection with Mycobacterium tuberculosis with no effect on disease progression.

Tuberculosis (TB) alone caused over a billion deaths in the last 200 years, making it one of the deadliest diseases to humankind. Understanding the immune mechanisms underlying protection or pathology in TB is key to uncover the much needed innovative approaches to tackle TB. The scavenger receptor cysteine-rich molecule CD5 antigen-like (CD5L) has been associated with TB, but whether and how CD5L shapes the immune response during the course of disease remains poorly understood. Here, we show an upregulation of CD5L in circulation and at the site of infection in C57BL/6 Mycobacterium tuberculosis-infected mice. To investigate the role of CD5L in TB, we studied the progression of M. tuberculosis aerosol infection in a recently described genetically engineered mouse model lacking CD5L. Despite the increase of CD5L during infection of wild-type mice, absence of CD5L did not impact bacterial burden, histopathology or survival of infected mice. Absence of CD5L associated with a modest increase in the numbers of CD4+ T cells and the expression of IFN-γ in the lungs of infected mice, with no major effect in overall immune cell dynamics. Collectively, this study confirms CD5L as a potential diagnostic biomarker to TB, showing no discernible impact on the outcome of the infection.

LAIR1 prevents excess inflammatory tissue damage in S. aureus skin infection and Cutaneous T-cell Lymphoma.

Patients with cutaneous T cell lymphoma (CTCL) experience high morbidity and mortality due to S. aureus skin infections and sepsis, but the causative immune defect is unclear. We previously identified high levels of LAIR2, a decoy protein for the inhibitory receptor LAIR1, in advanced CTCL. Mice do not have a LAIR2 homolog, so we used Lair1 knock-out (KO) mice to model LAIR2 overexpression. In a model of subcutaneous S. aureus skin infection, Lair1 KO mice had significantly larger abscesses and areas of dermonecrosis compared to WT. Lair1 KO exhibited a pattern of increased inflammatory responses in infection and sterile immune stimulation, including increased production of proinflammatory cytokines and myeloid chemokines, neutrophil ROS, and collagen/ECM remodeling pathways. Notably, Lair1 KO infected skin had a similar bacterial burden and neutrophils and monocytes had equivalent S. aureus phagocytosis compared to WT. These findings support a model in which lack of LAIR1 signaling causes an excessive inflammatory response that does not improve infection control. CTCL skin lesions harbored similar patterns of increased expression in cytokine and collagen/ECM remodeling pathways, suggesting that high levels of LAIR2 in CTCL recapitulates Lair1 KO, causing inflammatory tissue damage and compromising host defense against S. aureus infection.

Enhanced antibacterial properties of enteric glial cells attenuate intestinal inflammation through the GABABR-mediated autophagy pathway

Enterotoxigenic Escherichia coli (ETEC) infection is a severe threat to global public health because of its high morbidity and mortality among children and infants. Enteric glial cells (EGCs) are involved in host–bacteria communication. However, the mechanisms through which EGCs interact with ETEC remain unclear. We attempted to assess whether γ-aminobutyric acid type B receptor (GABABR) activation participated in EGC autophagy during Escherichia coli K88 (ETECK88) infection. Alterations in autophagy and EGC activity were observed in the intestines of the ETECK88-infected mice, and similar results were obtained from experiments in which the EGCs were directly infected with ETECK88. EGC pretreatment with specific autophagy agonists significantly decreased the inflammatory response and bacterial burden, whereas pretreatment with inhibitors had the opposite effect. Interestingly, in EGCs, GABABR activation notably increased Beclin 1 and LC3 levels and autophagosome and autolysosome numbers, thus promoting autophagy activation and enhancing antimicrobial responses against ETECK88 infection. Furthermore, GABABR defense was mediated via myeloid differentiation factor 88 (MyD88) signaling in EGCs, which was proven to be based on the inhibition or overexpression of MyD88. Notably, comparable results of GABABR activation in vivo were observed in response to ETECK88, implicating this as a defense mechanism that reinforced antibacterial activity to alleviate intestinal inflammation in mice. Our study revealed previously unappreciated roles for GABABR in linking EGC antibacterial autophagy to strengthen host defense against ETECK88 infection, thus identifying GABABR as an important target for the treatment of infective enteritis.

Immunostimulatory nucleic acid nanoparticles (NANPs) augment protective osteoblast and osteoclast type I interferon responses to Staphylococcus aureus

Recalcitrant staphylococcal osteomyelitis may be due, in part, to the ability of Staphylococcus aureus to invade bone cells. However, osteoclasts and osteoblasts are now recognized to shape host responses to bacterial infection and we have recently described their ability to produce IFN-β following S. aureus infection and limit intracellular bacterial survival/propagation. Here, we have investigated the ability of novel, rationally designed, nucleic acid nanoparticles (NANPs) to induce the production of immune mediators, including IFN-β, following introduction into bone cells. We demonstrate the successful delivery of representative NANPs into osteoblasts and osteoclasts via endosomal trafficking when complexed with lipid-based carriers. Their delivery was found to differentially induce immune responses according to their composition and architecture via discrete cytosolic pattern recognition receptors. Finally, the utility of this nanoparticle technology was supported by the demonstration that immunostimulatory NANPs augment IFN-β production by S. aureus infected bone cells and reduce intracellular bacterial burden.

Dysregulation of stress erythropoiesis and enhanced susceptibility to Salmonella Typhimurium infection in AhR-deficient mice.

By acting as an environmental sensor, the ligand-induced transcription factor aryl hydrocarbon receptor (AhR) regulates acute innate and adaptive immune responses against pathogens. Here, we analyzed the function of AhR in a model for chronic systemic infection with attenuated Salmonella Typhimurium (STM). WT and AhR-deficient mice were infected with the attenuated STM strain TAS2010 and analyzed for bacterial burden, host defense functions and inflammatory stress erythropoiesis. AhR-deficient mice were highly susceptible to TAS2010 infection compared with WT mice demonstrated by reduced bacterial clearance and increased mortality. STM infection resulted in macrocytic anemia and enhanced splenomegaly along with destruction of the splenic architecture in AhR-deficient mice. In addition, AhR-deficient mice displayed a major expansion of splenic immature red blood cells, indicative of infection-induced stress erythropoiesis. Elevated serum levels of erythropoietin and interleukin-6 upon infection as well as increased numbers of splenic stress erythroid progenitors already in steady state probably drive this effect and might cause the alterations in splenic immune cell compartments, thereby preventing an effective host defense against STM in AhR-deficient mice. AhR-deficient mice fail to clear chronic TAS2010 infection due to enhanced stress erythropoiesis in the spleen and accompanying destruction of the splenic architecture.

Therapeutic Modulation of Arginase with nor-NOHA Alters Immune Responses in Experimental Mouse Models of Pulmonary Tuberculosis including in the Setting of Human Immunodeficiency Virus (HIV) Co-Infection.

L-arginine metabolism is strongly linked with immunity to mycobacteria, primarily through the antimicrobial activity of nitric oxide (NO). The potential to modulate tuberculosis (TB) outcomes through interventions that target L-arginine pathways are limited by an incomplete understanding of mechanisms and inadequate in vivo modeling. These gaps in knowledge are compounded for HIV and Mtb co-infections, where activation of arginase-1 due to HIV infection may promote survival and replication of both Mtb and HIV. We utilized in vitro and in vivo systems to determine how arginase inhibition using Nω-hydroxy-nor-L-arginine (nor-NOHA) alters L-arginine pathway metabolism relative to immune responses and disease outcomes following Mtb infection. Treatment with nor-NOHA polarized murine macrophages (RAW 264.7) towards M1 phenotype, increased NO, and reduced Mtb in RAW macrophages. In Balb/c mice, nor-NOHA reduced pulmonary arginase and increased the antimicrobial metabolite spermine in association with a trend towards reduced Mtb CFU in lung. In humanized immune system (HIS) mice, HIV infection increased plasma arginase and heightened the pulmonary arginase response to Mtb. Treatment with nor-NOHA increased cytokine responses to Mtb and Mtb/HIV in lung tissue but did not significantly alter bacterial burden or viral load. Our results suggest that L-arginine pathway modulators may have potential as host-directed therapies to augment antibiotics in TB chemotherapy.

Deficiency in non-classical major histocompatibility class II-like molecule, H2-O confers protection against Staphylococcus aureus in mice.

Staphylococcus aureus is a human-adapted pathogen that replicates by asymptomatically colonizing its host. S. aureus is also the causative agent of purulent skin and soft tissue infections as well as bloodstream infections that result in the metastatic seeding of abscess lesions in all organ tissues. Prolonged colonization, infection, disease relapse, and recurrence point to the versatile capacity of S. aureus to bypass innate and adaptive immune defenses as well as the notion that some hosts fail to generate protective immune responses. Here, we find a genetic trait that provides protection against this pathogen. Mice lacking functional H2-O, the equivalent of human HLA-DO, inoculated with a mouse-adapted strain of S. aureus, efficiently decolonize the pathogen. Further, these decolonized animals resist subsequent bloodstream challenge with methicillin-resistant S. aureus. A genetic approach demonstrates that T-cell dependent B cell responses are required to control S. aureus colonization and infection in H2-O-deficient mice. Reduced bacterial burdens in these animals correlate with increased titers and enhanced phagocytic activity of S. aureus-specific antibodies. H2-O negatively regulates the loading of high affinity peptides on major histocompatibility class II (MHC-II) molecules. Thus, we hypothesize that immune responses against S. aureus are derepressed in mice lacking H2-O because more high affinity peptides are presented by MHC-II. We speculate that loss-of-function HLA-DO alleles may similarly control S. aureus replication in humans.

Vaccination with Mincle agonist UM-1098 and mycobacterial antigens induces protective Th1 and Th17 responses

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the top infectious killers in the world. The only licensed vaccine against TB, Bacille Calmette-Guérin (BCG), provides variable protection against pulmonary TB, especially in adults. Hence, novel TB vaccine approaches are urgently needed. Both Th1 and Th17 responses are necessary for protection against TB, yet effective adjuvants and vaccine delivery systems for inducing robust Th1 and Th17 immunity are lacking. Herein we describe a synthetic Mincle agonist, UM-1098, and a silica nanoparticle delivery system that drives Th1/Th17 responses to Mtb antigens. Stimulation of human peripheral blood mononuclear cells (hPBMCs) with UM-1098 induced high levels of Th17 polarizing cytokines IL-6, IL-1β, IL-23 as well as IL-12p70, IL-4 and TNF-α in vitro. PBMCs from both C57BL/6 and BALB/c mice responded with a similar cytokine pattern in vitro and in vivo. Importantly, intramuscular (I.M.) vaccination with UM-1098-adjuvanted TB antigen M72 resulted in significantly higher antigen-specific IFN-γ and IL-17A levels in C57BL/6 wt mice than Mincle KO mice. Vaccination of C57BL/6 wt mice with immunodominant Mtb antigens ESAT6/Ag85B or M72 resulted in predominantly Th1 and Th17 responses and induced antigen-specific serum antibodies. Notably, in a virulent Mtb challenge model, vaccination with UM-1098 adjuvanted ESAT6/Ag85B or M72 significantly reduced lung bacterial burden when compared with unvaccinated mice and protection occurred in the absence of pulmonary inflammation. These data demonstrate that the synthetic Mincle agonist UM-1098 induces strong Th1 and Th17 immunity after vaccination with Mtb antigens and provides protection against Mtb infection in mice.