Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia.

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury.

Pneumolysin suppresses the initial macrophage pro-inflammatory response to Streptococcus pneumoniae.

Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection.

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection.

Maintained partial protection against Streptococcus pneumoniae despite B-cell depletion in mice vaccinated with a pneumococcal glycoconjugate vaccine.

OBJECTIVES: Anti-CD20 monoclonal antibody therapy rapidly depletes > 95% of CD20+ B cells from the circulation. B-cell depletion is an effective treatment for autoimmune disease and B-cell malignancies but also increases the risk of respiratory tract infections. This effect on adaptive immunity could be countered by vaccination. We have used mouse models to investigate the effects of B-cell depletion on pneumococcal vaccination, including protection against infection and timing of vaccination in relation to B-cell depletion. METHODS: C57BL/6 female mice were B-cell depleted using anti-CD20 antibody and immunized with two doses of Prevnar-13 vaccine either before or after anti-CD20 treatment. B-cell repertoire and Streptococcus pneumoniae-specific IgG levels were measured using whole-cell ELISA and flow cytometry antibody-binding assay. Protection induced by vaccination was assessed by challenging the mice using a S. pneumoniae pneumonia model. RESULTS: Antibody responses to S. pneumoniae were largely preserved in mice B-cell depleted after vaccination resulting in full protection against pneumococcal infections. In contrast, mice vaccinated with Prevnar-13 while B cells were depleted (with > 90% reduction in B-cell numbers) had decreased circulating anti-S. pneumoniae IgG and IgM levels (measured using ELISA and flow cytometry antibody binding assays). However, some antibody responses were maintained, and, although vaccine-induced protection against S. pneumoniae infection was impaired, septicaemia was still prevented in 50% of challenged mice. CONCLUSIONS: This study showed that although vaccine efficacy during periods of profound B-cell depletion was impaired some protective efficacy was preserved, suggesting that vaccination remains beneficial.

The Influence of B Cell Depletion Therapy on Naturally Acquired Immunity to Streptococcus pneumoniae.

The anti-CD20 antibody Rituximab to deplete CD20+ B cells is an effective treatment for rheumatoid arthritis and B cell malignancies, but is associated with an increased incidence of respiratory infections. Using mouse models we have investigated the consequences of B cell depletion on natural and acquired humoral immunity to Streptococcus pneumoniae. B cell depletion of naïve C57Bl/6 mice reduced natural IgM recognition of S. pneumoniae, but did not increase susceptibility to S. pneumoniae pneumonia. ELISA and flow cytometry assays demonstrated significantly reduced IgG and IgM recognition of S. pneumoniae in sera from mice treated with B cell depletion prior to S. pneumoniae nasopharyngeal colonization compared to untreated mice. Colonization induced antibody responses to protein rather than capsular antigen, and when measured using a protein array B cell depletion prior to colonization reduced serum levels of IgG to several protein antigens. However, B cell depleted S. pneumoniae colonized mice were still partially protected against both lung infection and septicemia when challenged with S. pneumoniae after reconstitution of their B cells. These data indicate that although B cell depletion markedly impairs antibody recognition of S. pneumoniae in colonized mice, some protective immunity is maintained, perhaps mediated by cellular immunity.

Relative Contributions of Extracellular and Internalized Bacteria to Early Macrophage Proinflammatory Responses to Streptococcus pneumoniae.

Both intracellular immune sensing and extracellular innate immune sensing have been implicated in initiating macrophage proinflammatory cytokine responses to Streptococcus pneumoniae The S. pneumoniae capsule, a major virulence determinant, prevents phagocytosis, and we hypothesized that this would reduce activation of host innate inflammatory responses by preventing activation of intracellular proinflammatory signaling pathways. We investigated this hypothesis in human monocyte-derived macrophages stimulated with encapsulated or isogenic unencapsulated mutant S. pneumoniae Unexpectedly, despite strongly inhibiting bacterial internalization, the capsule resulted in enhanced inflammatory cytokine production by macrophages infected with S. pneumoniae Experiments using purified capsule material and a Streptococcus mitis mutant expressing an S. pneumoniae serotype 4 capsule indicated these differences required whole bacteria and were not due to proinflammatory effects of the capsule itself. Transcriptional profiling demonstrated relatively few differences in macrophage gene expression profiles between infections with encapsulated S. pneumoniae and those with unencapsulated S. pneumoniae, largely limited to reduced expression of proinflammatory genes in response to unencapsulated bacteria, predicted to be due to reduced activation of the NF-κB family of transcription factors. Blocking S. pneumoniae internalization using cytochalasin D had minimal effects on the inflammatory response to S. pneumoniae Experiments using murine macrophages indicated that the affected genes were dependent on Toll-like receptor 2 (TLR2) activation, although not through direct stimulation of TLR2 by capsule polysaccharide. Our data demonstrate that the early macrophage proinflammatory response to S. pneumoniae is mainly dependent on extracellular bacteria and reveal an unexpected proinflammatory effect of encapsulated S. pneumoniae that could contribute to disease pathogenesis.IMPORTANCE Multiple extra- and intracellular innate immune receptors have been identified that recognize Streptococcus pneumoniae, but the relative contributions of intra- versus extracellular bacteria to the inflammatory response were unknown. We have shown that intracellular S. pneumoniae contributes surprisingly little to the inflammatory responses, with production of important proinflammatory cytokines largely dependent on extracellular bacteria. Furthermore, although we expected the S. pneumoniae polysaccharide capsule to block activation of the host immune system by reducing bacterial internalization and therefore activation of intracellular innate immune receptors, there was an increased inflammatory response to encapsulated compared to unencapsulated bacteria, which is likely to contribute to disease pathogenesis.

Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.

Bacterial septicaemia is a major cause of mortality, but its pathogenesis remains poorly understood. In experimental pneumococcal murine intravenous infection, an initial reduction of bacteria in the blood is followed hours later by a fatal septicaemia. These events represent a population bottleneck driven by efficient clearance of pneumococci by splenic macrophages and neutrophils, but as we show in this study, accompanied by occasional intracellular replication of bacteria that are taken up by a subset of CD169+ splenic macrophages. In this model, proliferation of these sequestered bacteria provides a reservoir for dissemination of pneumococci into the bloodstream, as demonstrated by its prevention using an anti-CD169 monoclonal antibody treatment. Intracellular replication of pneumococci within CD169+ splenic macrophages was also observed in an ex vivo porcine spleen, where the microanatomy is comparable with humans. We also showed that macrolides, which effectively penetrate macrophages, prevented septicaemia, whereas beta-lactams, with inefficient intracellular penetration, failed to prevent dissemination to the blood. Our findings define a shift in our understanding of the pneumococcus from an exclusively extracellular pathogen to one with an intracellular phase. These findings open the door to the development of treatments that target this early, previously unrecognized intracellular phase of bacterial sepsis.

LytM proteins play a crucial role in cell separation, outer membrane composition, and pathogenesis in nontypeable Haemophilus influenzae.

UNLABELLED: LytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable Haemophilus influenzae (NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the Escherichia coli proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI ΔyebA and ΔnlpD deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the ΔenvC deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of ß-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI envC defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition. IMPORTANCE: Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that colonizes the human nasopharynx and can cause serious infections in children (acute otitis media) and adults (chronic obstructive pulmonary disease). Several virulence factors are well studied, but the complete scenario of NTHI pathogenesis is still unclear. We identified and characterized three NTHI LytM factors homologous to the Escherichia coli LytM proteins. Although LytM factors are reported to play a crucial role in the cell division process, in NTHI they are also involved in other bacterial functions. In particular, YebA and NlpD are fundamental for membrane stability: indeed, their absence causes an increased release of outer membrane vesicles (OMVs). On the other hand, our data suggest that EnvC could directly or indirectly affect peptidoglycan permeability and consequently, bacterial periplasmic and outer membrane protein distribution. Interestingly, by modulating the surface composition of virulence determinants, EnvC also has an impact on NTHI pathogenesis.