CD8+ T cells are necessary for improved sepsis survival induced by CD28 agonism in immunologically experienced mice.

Introduction: A hallmark of T cell dysregulation during sepsis is the downregulation of costimulatory molecules. CD28 is one of T cell costimulatory molecules significantly altered on memory T cells during sepsis. We recently showed that treatment with a αCD28 agonist in septic immunologically experienced mice led to improved survival. Therefore, here we aimed to identify the cell subset(s) necessary for the survival benefit observed in the context of CD28 agonism, and to further investigate the mechanism by which CD28 agonism improves sepsis survival in immunologically experienced mice. Methods: Mice received specific pathogen inoculation to generate memory T cell populations similar in frequency to that of adult humans. Once these infections were cleared and the T cell response had transitioned to the memory phase, animals were rendered septic via cecal ligation and puncture in the presence or absence of an agonistic anti-CD28 mAb. Results: Results demonstrated that CD8+ T cells, and not bulk CD4+ T cells or CD25+ regulatory T cells, were necessary for the survival benefit observed in CD28 agonist-treated septic immunologically experienced mice. Upon examination of these CD8+ T cells, we found that CD28 agonism in septic immunologically experienced mice was associated with an increase in Foxp3+ CD8+ T cells as compared to vehicle-treated controls. When CD8+ T cells were depleted in septic immunologically experienced mice in the setting of CD28 agonism, a significant increase in levels of inflammatory cytokines in the blood was observed. Discussion: Taken together, these results indicate that CD28 agonism in immunologically experienced mice effectively suppresses inflammation via a CD8+-dependent mechanism to decrease mortality during sepsis.

Claudin-2 upregulation enhances intestinal permeability, immune activation, dysbiosis, and mortality in sepsis.

Intestinal epithelial expression of the tight junction protein claudin-2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin-2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin-2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin-2 knockout (KO) and wild-type (WT) mice. Sepsis-induced increases in pore pathway permeability were prevented by claudin-2 KO. Moreover, claudin-2 deletion reduced interleukin-17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin-2 KO mice. Most strikingly, claudin-2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin-2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin-2 KO mice 24 h after sepsis. Claudin-2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin-2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin-2 may be an attractive therapeutic target in septic patients.

Effect of an initial specimen diversion device on blood-culture contamination rates and vancomycin usage: A quasi-experimental study.

Initial specimen diversion devices (ISDDs) are a potential solution for reducing blood-culture contamination rates. We report the implementation of an ISDD associated with a sustained reduction in blood-culture contamination rates for >18 months after implementation. We did not observe a clinically significant reduction in inpatient vancomycin usage.

Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

Translocation of gut commensal bacteria to the brain.

The gut-brain axis, a bidirectional signaling network between the intestine and the central nervous system, is crucial to the regulation of host physiology and inflammation. Recent advances suggest a strong correlation between gut dysbiosis and neurological diseases, however, relatively little is known about how gut bacteria impact the brain. Here, we reveal that gut commensal bacteria can translocate directly to the brain when mice are fed an altered diet that causes dysbiosis and intestinal permeability, and that this also occurs without diet alteration in distinct murine models of neurological disease. The bacteria were not found in other systemic sites or the blood, but were detected in the vagus nerve. Unilateral cervical vagotomy significantly reduced the number of bacteria in the brain, implicating the vagus nerve as a conduit for translocation. The presence of bacteria in the brain correlated with microglial activation, a marker of neuroinflammation, and with neural protein aggregation, a hallmark of several neurodegenerative diseases. In at least one model, the presence of bacteria in the brain was reversible as a switch from high-fat to standard diet resulted in amelioration of intestinal permeability, led to a gradual loss of detectable bacteria in the brain, and reduced the number of neural protein aggregates. Further, in murine models of Alzheimer's disease, Parkinson's disease, and autism spectrum disorder, we observed gut dysbiosis, gut leakiness, bacterial translocation to the brain, and microglial activation. These data reveal a commensal bacterial translocation axis to the brain in models of diverse neurological diseases.

CHRONIC ETHANOL USE WORSENS GUT PERMEABILITY AND ALTERS TIGHT JUNCTION EXPRESSION IN A MURINE SEPSIS MODEL.

ABSTRACT: Alcohol use disorder is associated with increased mortality in septic patients. Murine studies demonstrate that ethanol/sepsis is associated with changes in gut integrity. This study examined intestinal permeability after ethanol/sepsis and investigated mechanisms responsible for alterations in barrier function. Mice were randomized to drink either 20% ethanol or water for 12 weeks and then were subjected to either sham laparotomy or cecal ligation and puncture (CLP). Intestinal permeability was disproportionately increased in ethanol/septic mice via the pore, leak, and unrestricted pathways. Consistent with increased permeability in the leak pathway, jejunal myosin light chain (MLC) kinase (MLCK) expression and the ratio of phospho-MLC to total MLC were both increased in ethanol/CLP. Gut permeability was altered in MLCK -/- mice in water/CLP; however, permeability was not different between WT and MLCK -/- mice in ethanol/CLP. Similarly, jejunal IL-1ß levels were decreased while systemic IL-6 levels were increased in MLCK -/- mice in water/CLP but no differences were identified in ethanol/CLP. While we have previously shown that mortality is improved in MLCK -/- mice after water/CLP, mortality was significantly worse in MLCK -/- mice after ethanol/CLP. Consistent with an increase in the pore pathway, claudin 4 levels were also selectively decreased in ethanol/CLP WT mice. Furthermore, mRNA expression of jejunal TNF and IFN-γ were both significantly increased in ethanol/CLP. The frequency of CD4 + cells expressing TNF and IL-17A and the frequency of CD8 + cells expressing IFN-γ in Peyer's Patches were also increased in ethanol/CLP. Thus, there is an ethanol-specific worsening of gut barrier function after CLP that impacts all pathways of intestinal permeability, mediated, in part, via changes to the tight junction. Differences in the host response in the setting of chronic alcohol use may play a role in future precision medicine approaches toward the treatment of sepsis.

MYOSIN LIGHT CHAIN KINASE DELETION WORSENS LUNG PERMEABILITY AND INCREASES MORTALITY IN PNEUMONIA-INDUCED SEPSIS.

ABSTRACT: Increased epithelial permeability in sepsis is mediated via disruptions in tight junctions, which are closely associated with the perijunctional actin-myosin ring. Genetic deletion of myosin light chain kinase (MLCK) reverses sepsis-induced intestinal hyperpermeability and improves survival in a murine model of intra-abdominal sepsis. In an attempt to determine the generalizability of these findings, this study measured the impact of MLCK deletion on survival and potential associated mechanisms following pneumonia-induced sepsis. MLCK -/- and wild-type mice underwent intratracheal injection of Pseudomonas aeruginosa . Unexpectedly, survival was significantly worse in MLCK -/- mice than wild-type mice. This was associated with increased permeability to Evans blue dye in bronchoalveolar lavage fluid but not in tissue homogenate, suggesting increased alveolar epithelial leak. In addition, bacterial burden was increased in bronchoalveolar lavage fluid. Cytokine array using whole-lung homogenate demonstrated increases in multiple proinflammatory and anti-inflammatory cytokines in knockout mice. These local pulmonary changes were associated with systemic inflammation with increased serum levels of IL-6 and IL-10 and a marked increase in bacteremia in MLCK -/- mice. Increased numbers of both bulk and memory CD4 + T cells were identified in the spleens of knockout mice, with increased early and late activation. These results demonstrate that genetic deletion of MLCK unexpectedly increases mortality in pulmonary sepsis, associated with worsened alveolar epithelial leak and both local and systemic inflammation. This suggests that caution is required in targeting MLCK for therapeutic gain in sepsis.

The RIPK3 Scaffold Regulates Lung Inflammation During Pseudomonas Aeruginosa Pneumonia.

RIPK3 (receptor-interacting protein kinase 3) activity triggers cell death via necroptosis, whereas scaffold function supports protein binding and cytokine production. To determine if RIPK3 kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection, control mice and those with deletion or mutation of RIPK3 and associated signaling partners were subjected to Pseudomonas pneumonia and followed for survival or killed for biologic assays. Murine immune cells were studied in vitro for Pseudomonas-induced cytokine production and cell death, and RIPK3 binding interactions were blocked with the viral inhibitor M45. Human tissue effects were assayed by infecting airway epithelial cells with Pseudomonas and measuring cytokine production after siRNA inhibition of RIPK3. Deletion of RIPK3 reduced inflammation and decreased animal mortality after Pseudomonas pneumonia. RIPK3 kinase inactivation did neither. In cell culture, RIPK3 was dispensable for cell killing by Pseudomonas and instead drove cytokine production that required the RIPK3 scaffold domain but not kinase activity. Blocking the RIP homotypic interaction motif (RHIM) with M45 reduced the inflammatory response to infection in vitro. Similarly, siRNA knockdown of RIPK3 decreased infection-triggered inflammation in human airway epithelial cells. Thus, the RIPK3 scaffold drives deleterious pulmonary inflammation and mortality in a relevant clinical model of Pseudomonas pneumonia. This process is distinct from kinase-mediated necroptosis, requiring only the RIPK3 RHIM. Inhibition of RHIM signaling is a potential strategy to reduce lung inflammation during infection.

Junctional adhesion molecule-A deletion increases phagocytosis and improves survival in a murine model of sepsis.

Expression of the tight junction-associated protein junctional adhesion molecule-A (JAM-A) is increased in sepsis, although the significance of this is unknown. Here, we show that septic JAM-A -/- mice have increased gut permeability, yet paradoxically have decreased bacteremia and systemic TNF and IL-1ß expression. Survival is improved in JAM-A-/- mice. However, intestine-specific JAM-A-/- deletion does not alter mortality, suggesting that the mortality benefit conferred in mice lacking JAM-A is independent of the intestine. Septic JAM-A-/- mice have increased numbers of splenic CD44hiCD4+ T cells, decreased frequency of TNF+CD4+ cells, and elevated frequency of IL-2+CD4+ cells. Septic JAM-A-/- mice have increased numbers of B cells in mesenteric lymph nodes with elevated serum IgA and intraepithelial lymphocyte IgA production. JAM-A-/- × RAG-/- mice have improved survival compared with RAG-/- mice and identical mortality as WT mice. Gut neutrophil infiltration and neutrophil phagocytosis are increased in JAM-A-/- mice, while septic JAM-A-/- mice depleted of neutrophils lose their survival advantage. Therefore, increased bacterial clearance via neutrophils and an altered systemic inflammatory response with increased opsonizing IgA produced through the adaptive immune system results in improved survival in septic JAM-A-/- mice. JAM-A may be a therapeutic target in sepsis via immune mechanisms not related to its role in permeability.

Clinical Utilization of DiaSorin Molecular Polymerase Chain Reaction in Pneumocystis Pneumonia.

BACKGROUND: Pneumocystis jirovecii polymerase chain reaction (PCR) testing is a sensitive diagnostic tool but does not distinguish infection from colonization. Cycle threshold (CT) may correlate with fungal burden and could be considered in clinical decision making. Clinical use of PCR and significance of CT values have not previously been examined with the DiaSorin Molecular platform. METHODS: Retrospective review of P jirovecii PCR, CT values and clinical data from 18 months in a multihospital academic health system. The diagnostic performance of PCR with respect to pathology and correlation of CT with severity were examined. RESULTS: Ninety-nine of 1006 (9.8%) assays from 786 patients in 919 encounters were positive. Among 91 (9.9%) encounters in which P jirovecii pneumonia (PJP) was treated, 41 (45%) were influenced by positive PCR. Negative PCR influenced discontinuation of therapy in 35 cases. Sensitivity and specificity of PCR were 93% (95% CI, 68%-100%) and 94% (95% CI, 91%-96%) with respect to pathology. CT values from deep respiratory specimens were significantly different among treated patients (P = .04) and those with positive pathology results (P < .0001) compared to patients not treated and those with negative pathology, respectively, and was highly predictive of positive pathology results (area under the curve = 0.92). No significant difference was observed in comparisons based on indicators of disease severity. CONCLUSIONS: Pneumocystis jirovecii PCR was a highly impactful tool in the diagnosis and management of PJP, and use of CT values may have value in the treatment decision process in select cases. Further investigation in a prospective manner is needed.

Integrated evaluation of lung disease in single animals.

During infectious disease, pathogen load drives inflammation and immune response that together contribute to tissue injury often resulting in organ dysfunction. Pulmonary failure in SARS-CoV2-infected hospitalized COVID-19 patients is one such prominent example. Intervention strategies require characterization of the host-pathogen interaction by accurately assessing all of the above-mentioned disease parameters. To study infection in intact mammals, mice are often used as essential genetic models. Due to humane concerns, there is a constant unmet demand to develop studies that reduce the number of mice utilized while generating objective data. Here, we describe an integrated method of evaluating lung inflammation in mice infected with Pseudomonas aeruginosa or murine gammaherpesvirus (MHV)-68. This method conserves animal resources while permitting evaluation of disease mechanisms in both infection settings. Lungs from a single euthanized mouse were used for two purposes-biological assays to determine inflammation and infection load, as well as histology to evaluate tissue architecture. For this concurrent assessment of multiple parameters from a single euthanized mouse, we limit in-situ formalin fixation to the right lung of the cadaver. The unfixed left lung is collected immediately and divided into several segments for biological assays including determination of pathogen titer, assessment of infection-driven cytokine levels and appearance of cell death markers. In situ fixed right lung was then processed for histological determination of tissue injury and confirmation of infection-driven cell death patterns. This method reduces overall animal use and minimizes inter-animal variability that results from sacrificing different animals for different types of assays. The technique can be applied to any lung disease study in mice or other mammals.

Membrane Permeant Inhibitor of Myosin Light Chain Kinase Worsens Survival in Murine Polymicrobial Sepsis.

ABSTRACT: Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Genetic deletion of myosin light chain kinase (MLCK) reverses intestinal hyperpermeability and improves survival in a murine model of intra-abdominal sepsis. In an attempt to determine whether these findings could be translated using a more clinically relevant strategy, this study aimed to determine if pharmacologic inhibition of MLCK using the membrane permeant inhibitor of MLCK (PIK) improved gut barrier function and survival following sepsis. C57BL/6 mice underwent cecal ligation and puncture to induce sepsis and were then randomized to receive either PIK or vehicle. Unexpectedly, PIK significantly worsened 7-day survival following sepsis (24% vs. 62%). The three pathways of intestinal permeability were then interrogated by orally gavaging septic mice with creatinine (6Å), FD-4 (28Å), and rhodamine70 (120Å) and assaying their appearance in the bloodstream. PIK led to increased permeability in the leak pathway with higher levels of FD-4 in the bloodstream compared to septic mice given vehicle. In contrast, no differences were detected in the pore or unrestricted pathways of permeability. Examination of jejunal tight junctions for potential mechanisms underlying increased leak permeability revealed that mice that received PIK had increased phosphorylated MLC without alterations in occludin, ZO-1, or JAM-A. PIK administration was not associated with significant differences in systemic or peritoneal bacterial burden, cytokines, splenic or Peyer's Patches immune cells or intestinal integrity. These results demonstrate that pharmacologic inhibition of MLCK unexpectedly increases mortality, associated with worsened intestinal permeability through the leak pathway, and suggest caution is required in targeting the gut barrier as a potential therapy in sepsis.

Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States.

Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States.IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.

Antibiotic-Selected Gene Amplification Heightens Metal Resistance.

The increasing frequency of antibiotic resistance poses myriad challenges to modern medicine. Environmental survival of multidrug-resistant bacteria in health care facilities, including hospitals, creates reservoirs for transmission of these difficult to treat pathogens. To prevent bacterial colonization, these facilities deploy an array of infection control measures, including bactericidal metals on surfaces, as well as implanted devices. Although antibiotics are routinely used in these health care environments, it is unknown whether and how antibiotic exposure affects metal resistance. We identified a multidrug-resistant Enterobacter clinical isolate that displayed heteroresistance to the antibiotic colistin, where only a minor fraction of cells within the population resist the drug. When this isolate was grown in the presence of colistin, a 9-kb DNA region was duplicated in the surviving resistant subpopulation, but surprisingly, was not required for colistin heteroresistance. Instead, the amplified region included a three-gene locus (ncrABC) that conferred resistance to the bactericidal metal, nickel. ncrABC expression alone was sufficient to confer nickel resistance to E. coli K-12. Due to its selection for the colistin-resistant subpopulation harboring the duplicated 9-kb region that includes ncrABC, colistin treatment led to enhanced nickel resistance. Taken together, these data suggest that the use of antibiotics may inadvertently promote enhanced resistance to antimicrobial metals, with potentially profound implications for bacterial colonization and transmission in the health care environment.IMPORTANCE To inhibit bacterial transmission and infection, health care facilities use bactericidal metal coatings to prevent colonization of surfaces and implanted devices. In these environments, antibiotics are commonly used, but their effect on metal resistance is unclear. The data described here reveal that exposure of a human isolate of Enterobacter cloacae to a last-line antibiotic, colistin, resulted in a DNA amplification that does not confer antibiotic resistance but instead facilitates resistance to the toxic metal nickel. This highlights a novel aspect of antibiotic and metal interplay. Concerningly, these data suggest the use of antibiotics could in some cases promote bacterial survival and colonization in the health care environment and ultimately increase transmission and infection of patients.