Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis1,2. Here we examine the development and consequences of vascularisation of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model characterised by organised granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis2. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule VEGFA. Pharmacological inhibition of the VEGF pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergise with the first-line anti-tubercular antibiotic rifampicin as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations3. Our data suggest that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting TB therapy, particularly in extensively drug-resistant disease where current antibiotic regimens are largely ineffective.

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C-C motif chemokine receptor 2 (CCR2) is a major chemokine axis that recruits myeloid cells including monocytes and macrophages. Thus far, CCR2 mice have not been found to be susceptible to infection with Mycobacterium tuberculosis (Mtb). Here, using a prototype W-Beijing family lineage 2 Mtb strain, HN878, we show that CCR2 mice exhibit increased susceptibility to tuberculosis (TB). Following exposure to Mtb HN878, alveolar macrophages (AMs) are amongst the earliest cells infected. We show that AMs accumulate early in the airways following infection and express CCR2. During disease progression, CCR2-expressing AMs exit the airways and localize within the TB granulomas. RNA-sequencing of sorted airway and non-airway AMs from infected mice show distinct gene expression profiles, suggesting that upon exit from airways and localization within granulomas, AMs become classically activated. The absence of CCR2 cells specifically at the time of AM egress from the airways resulted in enhanced susceptibility to Mtb infection. Furthermore, infection with an Mtb HN878 mutant lacking phenolic glycolipid (PGL) expression still resulted in increased susceptibility in CCR2 mice. Together, these data show a novel role for CCR2 in protective immunity against clinically relevant Mtb infections.

SUMMARY Pneumonias caused by influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity and high mortality rate. Previously, we have shown that bacterial pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae ( Spn ) infection. We observe that IAV synergistically sensitizes lung epithelial cells for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacological induction of oxidative stress without virus sensitizes cells for PFT-mediated necroptosis. Antioxidant treatment or inhibition of necroptosis reduces disease severity during secondary bacterial infection. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identify necroptosis inhibition and antioxidant therapy as potential intervention strategies.

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Streptococcus pneumoniae (Spn) is a bacterial pathogen known to colonize the upper respiratory tract and cause serious opportunistic diseases such as pneumonia, bacteremia, sepsis and meningitis. As a consequence, millions of attributable deaths occur annually, especially among infants, the elderly and immunocompromised individuals. Although current vaccines, composed of purified pneumococcal polysaccharide in free form or conjugated to a protein carrier, are widely used and have been demonstrated to be effective in target groups, Spn has continued to colonize and cause life-threatening disease in susceptible populations. This lack of broad protection highlights the necessity of improving upon the current “gold standard” pneumococcal vaccines to increase protection both by decreasing colonization and reducing the incidence of sterile-site infections. Over the past century, most of the pneumococcal proteins that play an essential role in colonization and pathogenesis have been identified and characterized. Some of these proteins have the potential to serve as antigens in a multi-valent protein vaccine that confers capsule independent protection. This review seeks to summarize the benefits and limitations of the currently employed vaccine strategies, describes how leading candidate proteins contribute to pneumococcal disease development, and discusses the potential of these proteins as protective antigens – including as a hybrid construct.

RationalePneumonia caused by Influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity. Previously we have shown that pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae (Spn) infection.ObjectivesDetermine the impact of IAV infection on bacterial PFT-mediated lung epithelial cell (LEC) necroptosis. Determine the molecular basis for increased sensitivity and if inhibition of necroptosis or oxidative stress blocks IAV sensitization of LEC to PFT.MethodsMice and cells were challenged with IAV followed by Spn. Necroptosis was monitored by measuring cell death at fixed time points post-infection and immunofluorescent detection of necroptosis. Wildtype mice and LEC were treated with necroptosis inhibitors. Necroptosis effector molecule MLKL deficiency was tested for infection synergy. Oxidative damage to DNA and lipids as result of infection was measured in vitro and in vivo. Necroptosis and anti-oxidant therapy efficacy to reduce disease severity was tested in vivo.Measurements and Main ResultsIAV synergistically sensitized LEC for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacological induction of oxidative stress sans virus sensitized cells for PFT-mediated necroptosis. Necroptosis inhibition reduced disease severity during secondary bacterial infection.ConclusionsIAV-induced oxidative stress sensitizes LEC for PFT-mediated necroptosis. This is a new molecular explanation for severe influenza-associated bacterial infections. Necroptosis inhibitors are potential therapeutic strategies to reduce IAV-primed bacterial pneumonia severity.SummaryHere we demonstrate that Influenza A virus (IAV) infection synergistically sensitizes lung cells to bacterial pore-forming toxin (PFT)-mediated necroptosis. Moreover, this contributes to the severity of lung injury that is observed during co- and secondary infection with Streptococcus pneumoniae. IAV-induced oxidative stress was identified as a key factor contributing to cell sensitization and induction of oxidative stress sans virus was sufficient to synergistically enhance susceptibility to PFT-mediated killing. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identifies necroptosis inhibition and antioxidant therapy as potential intervention strategies.

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