Interleukin-33 improves local immunity during Gram-negative pneumonia by a combined effect on neutrophils and inflammatory monocytes.

Pneumonia represents a major health care burden and Gram-negative bacteria provide an increasing therapeutic challenge at least in part through the emergence of multidrug-resistant strains. IL-33 is a multifunctional cytokine belonging to the IL-1 family that can affect many different cell types. We sought here to determine the effect of recombinant IL-33 on the host response during murine pneumonia caused by the common Gram-negative pathogen Klebsiella pneumoniae. IL-33 pretreatment prolonged survival for more than 1 day during lethal airway infection and decreased bacterial loads at the primary site of infection and distant organs. Postponed treatment with IL-33 (3 h) also reduced bacterial growth and dissemination. IL-33-mediated protection was not observed in mice deficient for the IL-33 receptor component IL-1 receptor-like 1. IL-33 induced a brisk type 2 response, characterized by recruitment of type 2 innate lymphoid cells to the lungs and enhanced release of IL-5 and IL-13. However, neither absence of innate lymphoid cells or IL-13, nor blocking of IL-5 impacted on IL-33 effects in mice infected with Klebsiella. Likewise, IL-33 remained effective in reducing bacterial loads in mice lacking B, T, and natural killer T cells. Experiments using antibody-mediated cell depletion indicated that neutrophils and inflammatory monocytes were of importance for antibacterial defense. The capacity of IL-33 to restrict bacterial growth in the lungs was strongly reduced in mice depleted of both neutrophils and inflammatory monocytes, but not in mice selectively depleted of either one of these cell types. These results suggest that IL-33 boosts host defense during bacterial pneumonia by a combined effect on neutrophils and inflammatory monocytes. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Neutrophil extracellular traps in patients with pulmonary tuberculosis.

Tuberculosis is a devastating infectious disease causing many deaths worldwide. Recent investigations have implicated neutrophil extracellular traps (NETs) in the host response to tuberculosis. The aim of the current study was to obtain evidence for NETs release in the circulation during human tuberculosis. For this we measured the plasma concentrations of nucleosomes in conjunction with neutrophil elastase, in 64 patients with active pulmonary tuberculosis and 32 healthy controls. Patients with active tuberculosis had elevated plasma levels of nucleosomes and elastase when compared with local healthy blood donors. Furthermore nucleosome and elastase levels showed a positive correlation. These findings provide the first evidence for the release of NETs in the circulation of patients with active pulmonary tuberculosis.

Granzymes A and B Regulate the Local Inflammatory Response during Klebsiella pneumoniae Pneumonia.

Klebsiella pneumoniae is a common cause of hospital-acquired pneumonia. Granzymes (gzms), mainly found in cytotoxic lymphocytes, have been implicated as mediators of infection and inflammation. We here sought to investigate the role of gzmA and gzmB in the host response to K. pneumoniae-induced airway infection and sepsis. For this purpose, pneumonia was induced in wild-type (WT) and gzmA-deficient (gzmA-/-), gzmB-/- and gzmAxB-/- mice by intranasal infection with K. pneumoniae. In WT mice, gzmA and gzmB were mainly expressed by natural killer cells. Pneumonia was associated with reduced intracellular gzmA and increased intracellular gzmB levels. Gzm deficiency had little impact on antibacterial defence: gzmA-/- and gzmAxB-/- mice transiently showed modestly higher bacterial loads in the lungs but not in distant organs. GzmB-/- and, to a larger extent, gzmAxB-/- mice displayed transiently increased lung inflammation, reflected in the semi-quantitative histology scores and levels of pro-inflammatory cytokines and chemokines. Most differences between gzm-deficient and WT mice had disappeared during late-stage pneumonia. Gzm deficiency did not impact on distant organ injury or survival. These results suggest that gzmA and gzmB partly regulate local inflammation during early pneumonia but eventually play an insignificant role during pneumosepsis by the common human pathogen K. pneumoniae.

Increased intra- and extracellular granzyme expression in patients with tuberculosis.

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8(+) T, CD4(+) T and CD56(+) T, but not NK, cells, and of gzmB in CD8(+) T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis.

Modular Transcriptional Networks of the Host Pulmonary Response during Early and Late Pneumococcal Pneumonia.

Streptococcus pneumoniae (Spneu) remains the most lethal bacterial pathogen and the dominant agent of community-acquired pneumonia. Treatment has perennially focused on the use of antibiotics, albeit scrutinized due to the occurrence of antibiotic-resistant Spneu strains. Immunomodulatory strategies have emerged as potential treatment options. Although promising, immunomodulation can lead to improper tissue functions either at steady state or upon infectious challenge. This argues for the availability of tools to enable a detailed assessment of whole pulmonary functions during the course of infection, not only those functions biased to the defense response. Thus, through the use of an unbiased tissue microarray and bioinformatics approach, we aimed to construct a comprehensive map of whole-lung transcriptional activity and cellular pathways during the course of pneumococcal pneumonia. We performed genome-wide transcriptional analysis of whole lungs before and 6 and 48 h after Spneu infection in mice. The 4,000 most variable transcripts across all samples were used to assemble a gene coexpression network comprising 13 intercorrelating modules (clusters of genes). Fifty-four percent of this whole-lung transcriptional network was altered 6 and 48 h after Spneu infection. Canonical signaling pathway analysis uncovered known pathways imparting protection, including IL17A/IL17F signaling and previously undetected mechanisms that included lipid metabolism. Through in silico prediction of cell types, pathways were observed to enrich for distinct cell types such as a novel stromal cell lipid metabolism pathway. These cellular mechanisms were furthermore anchored at functional hub genes of cellular fate, differentiation, growth and transcription. Collectively, we provide a benchmark unsupervised map of whole-lung transcriptional relationships and cellular activity during early and late pneumococcal pneumonia.

Expression of inhibitory regulators of innate immunity in patients with active tuberculosis.

BACKGROUND: Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Toll-like-receptors (TLRs) are important for the recognition of the causative agent Mycobacterium tuberculosis. Negative regulation of TLRs is necessary to control deleterious inflammatory damage, but could provide a means of immune evasion by M. tuberculosis as well. METHODS: To obtain insight in the extent of expression of inhibitory regulators of immunity in patients with active TB, peripheral-blood-mononuclear-cells (PBMCs) and plasma were obtained from 54 TB patients and 29 healthy blood donors from Chittagong, Bangladesh. Bilateral alveolar macrophages were obtained from an infected versus a contralateral normal lung segment of 9 patients. Statistical analyses were performed using Mann-Whitney U and Wilcoxon matched pairs testing. Correlations were calculated using the Spearman rho test. RESULTS: PBMCs harvested from TB patients demonstrated increased mRNA expression of IL-1-receptor-associated-kinase-M, suppressor-of-cytokine-signalling-3 and Toll-interacting-protein. Flow cytometry revealed enhanced expression of IL-1-receptor-like-1 (ST2) on lymphocytes. Plasma soluble ST2 was elevated in patients with TB and correlated with established TB biomarkers, most strongly with soluble interleukin-2 receptor subunit α and interleukin-8. Alveolar macrophage mRNA expression of negative TLR regulators did not differ between the infected and contralateral lung side. CONCLUSION: These results show enhanced expression of distinct negative regulators of innate immunity in PBMCs of patients with TB and identify plasma soluble ST2 as a potential novel biomarker for TB disease activity.

Pulmonary tuberculosis induces a systemic hypercoagulable state.

OBJECTIVES: Human tuberculosis (TB) remains an important cause of death globally. Bangladesh is one of the most affected countries. We aimed to investigate the impact of pulmonary TB on pro- and anticoagulant mechanisms. METHODS: This prospective study was conducted in Chittagong, Bangladesh. We performed an in-depth analysis of coagulation activation and inhibition in plasma obtained from 64 patients with primary lung TB and 11 patients with recurrent lung TB and compared these with 37 healthy controls. Additionally, in nine patients coagulation activation was studied in bronchoalveolar lavage fluid (BALF) harvested from the site of infection and compared with BALF from a contralateral unaffected lung subsegment. RESULTS: Relative to uninfected controls, primary and recurrent TB were associated with a systemic net procoagulant state, as indicated by enhanced activation of coagulation (elevated plasma levels of thrombin-antithrombin complexes, D-dimer and fibrinogen) together with impaired anticoagulant mechanisms (reduced plasma levels of antithrombin, protein C activity, free protein S, and protein C inhibitor). Activation of coagulation did not correlate with plasma concentrations of established TB biomarkers. Coagulation activation could not be detected at the primary site of infection in a subset of TB patients. CONCLUSIONS: Pulmonary TB is associated with a systemic hypercoagulable state.

Single immunoglobulin interleukin-1 receptor-related molecule impairs host defense during pneumonia and sepsis caused by Streptococcus pneumoniae.

Streptococcus pneumoniae is a common cause of pneumonia and sepsis. Toll-like receptors (TLRs) play a pivotal role in the host defense against infection. In this study, we sought to determine the role of single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR a.k.a. TIR8), a negative regulator of TLR signaling, in pneumococcal pneumonia and sepsis. Wild-type and SIGIRR-deficient (sigirr-/-) mice were infected intranasally (to induce pneumonia) or intravenously (to induce primary sepsis) with S. pneumoniae and euthanized after 6, 24, or 48 h for analyses. Additionally, survival studies were performed. sigirr-/- mice showed delayed mortality during lethal pneumococcal pneumonia. Accordingly, sigirr-/- mice displayed lower bacterial loads in lungs and less dissemination of the infection 24 h after the induction of pneumonia. SIGIRR deficiency was associated with increased interstitial and perivascular inflammation in lung tissue early after infection, with no impact on neutrophil recruitment or cytokine production. sigirr-/- mice also demonstrated reduced bacterial burdens at multiple body sites during S. pneumoniae sepsis. sigirr-/- alveolar macrophages and neutrophils exhibited an increased capacity to phagocytose viable pneumococci. These results suggest that SIGIRR impairs the antibacterial host defense during pneumonia and sepsis caused by S. pneumoniae.

Role of interleukin 1 receptor like 1 (ST2) in gram-negative and gram-positive sepsis in mice.

BACKGROUND: Interleukin 1 receptor-like 1 (ST2) has been implicated as a negative regulator of Toll-like receptor signaling. We here sought to elucidate the role of ST2 in cytokine release and systemic infection caused by two common human sepsis pathogens, Streptococcus pneumoniae (gram-positive) and Klebsiella pneumoniae (gram-negative). METHODS: Whole blood leukocytes and splenocytes were harvested from ST2-deficient (st2) and wild-type (WT) mice and stimulated ex vivo with S. pneumoniae or K. pneumoniae. In addition, st2 and WT mice were infected intravenously with these bacteria, and bacterial loads and cytokine levels were measured in blood, spleens and lungs at 6, 24, and 48 h thereafter. RESULTS: Unexpectedly, st2 blood leukocytes and splenocytes produced lower levels of cytokines and chemokines than WT cells in response to either pathogen. In contrast, the in vivo role of ST2 during sepsis caused by these bacteria was limited, although at 6 and 24 h after infection with S. pneumoniae bacterial loads were lower in spleens of st2 mice. CONCLUSIONS: ST2 augments rather than inhibits cytokine release by blood leukocytes and splenocytes exposed to S. pneumoniae or K. pneumoniae, but plays a limited role in host defense during sepsis caused by these pathogens.

Limited anti-inflammatory role for interleukin-1 receptor like 1 (ST2) in the host response to murine postinfluenza pneumococcal pneumonia.

Interleukin-1 receptor like 1 (ST2) is a negative regulator of Toll-like receptor (TLR) signaling. TLRs are important for host defense during respiratory tract infections by both influenza and Streptococcus (S.) pneumoniae. Enhanced susceptibility to pneumococcal pneumonia is an important complication following influenza virus infection. We here sought to determine the role of ST2 in primary influenza A infection and secondary pneumococcal pneumonia. ST2 knockout (st2(-/-)) and wild-type (WT) mice were intranasally infected with influenza A virus; in some experiments mice were infected 2 weeks later with S. pneumoniae. Both mouse strains cleared the virus similarly during the first 14 days of influenza infection and had recovered their weights equally at day 14. Overall st2(-/-) mice tended to have a stronger pulmonary inflammatory response upon infection with influenza; especially 14 days after infection modest but statistically significant elevations were seen in lung IL-6, IL-1ß, KC, IL-10, and IL-33 concentrations and myeloperoxidase levels, indicative of enhanced neutrophil activity. Interestingly, bacterial lung loads were higher in st2(-/-) mice during the later stages of secondary pneumococcal pneumonia, which was associated with relatively increased lung IFN-γ levels. ST2 deficiency did not impact on gross lung pathology in either influenza or secondary S. pneumoniae pneumonia. These data show that ST2 plays a limited anti-inflammatory role during both primary influenza and postinfluenza pneumococcal pneumonia.

Differential roles of MyD88 and TRIF in hematopoietic and resident cells during murine gram-negative pneumonia.

BACKGROUND: Pneumonia is frequently caused by gram-negative pathogens, among which Klebsiella pneumoniae prominently features. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is important for an appropriate immune response during infection. TLR signaling can proceed via 2 distinct routes that are dependent on the adaptor proteins Myeloid differentiation primary response gene (88) (MyD88) and TIR-domain-containing adaptor-inducing interferon-ß (TRIF). The aim of the study was to determine the relative contribution of MyD88 and TRIF signaling in resident and hematopoietic cells to host defense during pneumonia. METHODS: Bone marrow chimeras of MyD88 deficient/wild type and TRIF mutant/wild type mice were created and infected with K. pneumoniae via the airways. RESULTS: MyD88 in both resident and hematopoietic cells contributed to survival and antibacterial defense in late-stage infection, whereas only TRIF in hematopoietic cells was protective. On the other hand, resident MyD88 and hematopoietic TRIF contributed to distant cellular injury. Resident MyD88 was pivotal for early chemokine release and neutrophil recruitment in the bronchoalveolar space. CONCLUSIONS: MyD88- and TRIF-dependent signaling has a differential contribution to host defense in different cell types that changes from early- to late-stage gram-negative pneumonia.

R-roscovitine reduces lung inflammation induced by lipoteichoic acid and Streptococcus pneumoniae.

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniae in vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.

Interleukin-1 receptor-associated kinase M-deficient mice demonstrate an improved host defense during Gram-negative pneumonia.

Pneumonia is a common cause of morbidity and mortality and the most frequent source of sepsis. Bacteria that try to invade normally sterile body sites are recognized by innate immune cells through pattern recognition receptors, among which toll-like receptors (TLRs) feature prominently. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK)-M is a proximal inhibitor of TLR signaling expressed by epithelial cells and macrophages in the lung. To determine the role of IRAK-M in host defense against bacterial pneumonia, IRAK-M-deficient (IRAK-M(-/-)) and normal wild-type (WT) mice were infected intranasally with Klebsiella pneumoniae. IRAK-M mRNA was upregulated in lungs of WT mice with Klebsiella pneumonia, and the absence of IRAK-M resulted in a strongly improved host defense as reflected by reduced bacterial growth in the lungs, diminished dissemination to distant body sites, less peripheral tissue injury and better survival rates. Although IRAK-M(-/-) alveolar macrophages displayed enhanced responsiveness toward intact K. pneumoniae and Klebsiella lipopolysaccharide (LPS) in vitro, IRAK-M(-/-) mice did not show increased cytokine or chemokine levels in their lungs after infection in vivo. The extent of lung inflammation was increased in IRAK-M(-/-) mice shortly after K. pneumoniae infection, as determined by semiquantitative scoring of specific components of the inflammatory response in lung tissue slides. These data indicate that IRAK-M impairs host defense during pneumonia caused by a common gram-negative respiratory pathogen.

Interleukin 1 receptor-associated kinase m impairs host defense during pneumococcal pneumonia.

BACKGROUND: Streptococcus pneumoniae is the most common causative organism in community-acquired pneumonia. Pneumococci that try to invade the lower airways are recognized by innate immune cells through pattern recognition receptors, including Toll-like receptors 2, 4, and 9. Interleukin 1 (IL-1) receptor-associated kinase (IRAK)-M is a proximal inhibitor of Toll-like receptor signaling. METHODS: To determine the role of IRAK-M in host defense during pneumococcal pneumonia, IRAK-M- deficient and wild-type mice were intranasally infected with S. pneumoniae. RESULTS: IRAK-M-deficient mice demonstrated a reduced lethality after infection with S. pneumoniae via the airways. Whereas bacterial burdens were similar in IRAK-M-deficient and wild-type mice early (3 hours) after infection, from 24 hours onward the number of pneumococci recovered from lungs and distant body sites were 10-100-fold lower in the former mouse strain. The diminished bacterial growth and dissemination in IRAK-M-deficient mice were preceded by an increased early influx of neutrophils into lung tissue and elevated pulmonary levels of IL-1ß and CXCL1. IRAK-M deficiency did not influence bacterial growth after intravenous administration of S. pneumoniae. CONCLUSIONS: These data suggest that IRAK-M impairs host defense during pneumococcal pneumonia at the primary site of infection at least in part by inhibiting the early immune response.