The role of IL-27 in susceptibility to post-influenza Staphylococcus aureus pneumonia

Background

Influenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection.

Methods

A murine model of influenza and Staphylococcus aureus infection was used to mimic human viral, bacterial co-infection. C57BL/6 wild-type, IL-27 receptor α knock-out, and IL-10 knock-out mice were infected with Influenza H1N1 (A/PR/8/34) or vehicle for 6 days followed by challenge with Staphylococcus aureus or vehicle for 24 hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined.

Results

IL-27 receptor α knock-out mice challenged with influenza A had increased morbidity compared to controls, but no change in viral burden. IL-27 receptor α knock-out mice infected with influenza displayed significantly decreased IL-10 production compared to wild-type. IL-27 receptor α knock-out mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls. Importantly, there were significantly increased Type 17 responses and decreased IL-10 production in IL-27 receptor α knock-out mice. Dual infected IL-10−/− mice had significantly less bacterial burden compared to dual infected WT mice.

Conclusions

These data reveal that IL-27 regulates enhanced susceptibility to S. aureus pneumonia following influenza infection, potentially through the induction of IL-10 and suppression of IL-17.     

One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies,Effector CD4+ T Cells,and IL-17A

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4⁺ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4⁺ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.     

Th17 cell plasticity towards a T-bet-dependent Th1 phenotype is required for bacterial control in Staphylococcus aureus infection

Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4⁺ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice.To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads.In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.     

A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

Due to the enormous capacity of Staphylococcus aureus to acquire antibiotic resistance, it becomes imperative to develop vaccines for decreasing the risk of its life-threatening infections. Peptidoglycan (PGN) is a conserved and major component of S. aureus cell wall. However, it has not been used as a vaccine candidate since it is a thymus-independent antigen. In this study, we synthesized a multiple antigenic peptide, named MAP27, which comprised four copies of a peptide that mimics the epitope of PGN. After immunization with MAP27 five times and boosting with heat-inactivated bacterium one time, anti-MAP27 serum bound directly to S. aureus or PGN. Immunization with MAP27 decreased the bacterial burden in organs of BALB/c mice and significantly prolonged their survival time after S. aureus lethal-challenge. The percentage of IFN-γ⁺CD3⁺ T cells and IL-17⁺CD4⁺ T cells in spleen, as well as the levels of IFN-γ, IL-17A/F and CCL3 in spleen and lung, significantly increased in the MAP27-immunized mice after infection. Moreover, in vitro incubation of heat-inactivated S. aureus with splenocytes isolated from MAP27-immunized mice stimulated the production of IFN-γ and IL-17A/F. Our findings demonstrated that MAP27, as a thymus-dependent antigen, is efficient at eliciting T cell-mediated responses to protect mice from S. aureus infection. This study sheds light on a possible strategy to design vaccines against S. aureus.     

Early-Life Gut Bacteria Associate with IL-4?, IL-10? and IFN-γ Production at Two Years of Age

Microbial exposure early in life influences immune maturation and potentially also the development of immune-mediated disease. Here we studied early-life gut colonization in relation to cytokine responses at two years of age. Fecal samples were collected from infants during the first two months of life. DNA was extracted from the fecal samples and Bifidobacterium (B.) adolescentis, B. breve, B. bifidum, a group of lactobacilli (L. casei, L. paracasei and L. rhamnosus) as well as Staphylococcus (S.) aureus were detected with real time PCR. Peripheral mononuclear cells were stimulated with phytohaemagglutinin (PHA) and numbers of IL-4−, IL-10− and IFN-γ secreting cells were evaluated using ELISpot. We further stimulated peripheral blood mononuclear cells with bacterial supernatants in vitro and assessed the IL-4−, IL-10− and IFN-γ inducing capacity by flow cytometry and ELISA. Early S. aureus colonization associated with higher numbers of IL-4− (p = 0.022) and IL-10 (p = 0.016) producing cells at two years of age. In contrast to colonization with S. aureus alone, co-colonization with lactobacilli associated with suppression of IL-4− (p = 0.004), IL-10− (p = 0.004) and IFN-γ (p = 0.034) secreting cells. In vitro stimulations of mononuclear cells with bacterial supernatants supported a suppressive role of L. rhamnosus GG on S. aureus-induced cytokine responses. We demonstrate that the early gut colonization pattern associates with the PHA-induced cytokine profile at two years of age and our in vitro findings support that specific bacterial species influence the T helper cell subsets. This suggests that dysbiosis in the early microbiota may modulate the risk of developing inflammatory conditions like allergy.     

A Type VI Secretion System Encoding Locus Is Required for Bordetella bronchiseptica Immunomodulation and Persistence In Vivo

Type VI Secretion Systems (T6SSs) have been identified in numerous Gram-negative pathogens, but the lack of a natural host infection model has limited analysis of T6SS contributions to infection and pathogenesis. Here, we describe disruption of a gene within locus encoding a putative T6SS in Bordetella bronchiseptica strain RB50, a respiratory pathogen that circulates in a broad range of mammals, including humans, domestic animals, and mice. The 26 gene locus encoding the B. bronchiseptica T6SS contains apparent orthologs to all known core genes and possesses thirteen novel genes. By generating an in frame deletion of clpV, which encodes a putative ATPase required for some T6SS-dependent protein secretion, we observe that ClpV contributes to in vitro macrophage cytotoxicity while inducing several eukaryotic proteins associated with apoptosis. Additionally, ClpV is required for induction of IL-1β, IL-6, IL-17, and IL-10 production in J774 macrophages infected with RB50. During infections in wild type mice, we determined that ClpV contributes to altered cytokine production, increased pathology, delayed lower respiratory tract clearance, and long term nasal cavity persistence. Together, these results reveal a natural host infection system in which to interrogate T6SS contributions to immunomodulation and pathogenesis.     

Nasal Colonisation by Staphylococcus aureus Depends upon Clumping Factor B Binding to the Squamous Epithelial Cell Envelope Protein Loricrin

Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB⁺ S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor^−/−) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB⁻ mutant colonised wild-type mice less efficiently than the parental ClfB⁺ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB⁻ mutant in the Lor^−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor^−/− mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.     

Activation and IL-10 production of specific CD4+ T cells are regulated by IL-27 during chronic infection with Plasmodium chabaudi

IL-27, a regulatory cytokine, plays critical roles in the prevention of immunopathology during Plasmodium infection. We examined these roles in the immune responses against Plasmodium chabaudi infection using the Il-27ra^−/− mice. While IL-27 was expressed at high levels during the early phase of the infection, enhanced CD4⁺ T cell function and reduction in parasitemia were observed mainly during the chronic phase in the mutant mice. In mice infected with P. chabaudi and cured with drug, CD4⁺ T cells in the Il-27ra^−/− mice exhibited enhanced CD4⁺ T-cell responses, indicating the inhibitory role of IL-27 on the protective immune responses. To determine the role of IL-27 in detail, we performed CD4⁺ T-cell transfer experiments. The Il-27ra^−/− and Il27p28^−/− mice were first infected with P. chabaudi and then cured using drug treatment. Plasmodium-antigen primed CD4⁺ T cells were prepared from these mice and transferred into the recipient mice, followed by infection with the heterologous parasite P. berghei ANKA. Il-27ra^−/− CD4⁺ T cells in the infected recipient mice did not produce IL-10, indicating that IL-10 production by primed CD4⁺ T cells is IL-27 dependent. Il27p28^−/− CD4⁺ T cells that were primed in the absence of IL-27 exhibited enhanced recall responses during the challenge infection with P. berghei ANKA, implying that IL-27 receptor signaling during the primary infection affects recall responses in the long-term via the regulation of the memory CD4⁺ T cell generation. These features highlighted direct and time-transcending roles of IL-27 in the regulation of immune responses against chronic infection with Plasmodium parasites.     

Protective Role of Surfactant Protein D in Ocular Staphylococcus aureus Infection

Staphylococcus aureus is one of the most common pathogens causing keratitis. Surfactant protein D (SP-D) plays a critical role in host defense and innate immunity. In order to investigate the role of SP-D in ocular S. aureus infection, the eyes of wild-type (WT) and SP-D knockout (SP-D KO) C57BL/6 mice were infected with S. aureus (10⁷ CFU/eye) in the presence and absence of cysteine protease inhibitor(E₆₄).Bacterial counts in the ocular surface were examined 3, 6, 12, 24 hrs after infection. Bacterial phagocytosis by neutrophils and bacterial invasion in ocular epithelial cells were evaluated quantitatively. S. aureus-induced ocular injury was determined with corneal fluorescein staining. The results demonstrated that SP-D is expressed in ocular surface epithelium and the lacrimal gland; WT mice had increased clearance of S. aureus from the ocular surface (p<0.05) and reduced ocular injury compared with SP-D KO mice. The protective effects of SP-D include increased bacterial phagocytosis by neutrophils (p<0.05) and decreased bacterial invasion into epithelial cells (p<0.05) in WT mice compared to in SP-D KO mice. In the presence of inhibitor (E₆₄), WT mice showed enhanced bacterial clearance (p<0.05) and reduced ocular injury compared to absent E₆₄ while SP-D KO mice did not. Collectively, we concluded that SP-D protects the ocular surface from S. aureus infection but cysteine protease impairs SP-D function in this murine model, and that cysteine protease inhibitor may be a potential therapeutic agent in S. aureus keratitis.     

Branched chain fatty acid synthesis drives tissue-specific innate immune response and infection dynamics of Staphylococcus aureus

Fatty acid-derived acyl chains of phospholipids and lipoproteins are central to bacterial membrane fluidity and lipoprotein function. Though it can incorporate exogenous unsaturated fatty acids (UFA), Staphylococcus aureus synthesizes branched chain fatty acids (BCFA), not UFA, to modulate or increase membrane fluidity. However, both endogenous BCFA and exogenous UFA can be attached to bacterial lipoproteins. Furthermore, S. aureus membrane lipid content varies based upon the amount of exogenous lipid in the environment. Thus far, the relevance of acyl chain diversity within the S. aureus cell envelope is limited to the observation that attachment of UFA to lipoproteins enhances cytokine secretion by cell lines in a TLR2-dependent manner. Here, we leveraged a BCFA auxotroph of S. aureus and determined that driving UFA incorporation disrupted infection dynamics and increased cytokine production in the liver during systemic infection of mice. In contrast, infection of TLR2-deficient mice restored inflammatory cytokines and bacterial burden to wildtype levels, linking the shift in acyl chain composition toward UFA to detrimental immune activation in vivo. In in vitro studies, bacterial lipoproteins isolated from UFA-supplemented cultures were resistant to lipase-mediated ester hydrolysis and exhibited heightened TLR2-dependent innate cell activation, whereas lipoproteins with BCFA esters were completely inactivated after lipase treatment. These results suggest that de novo synthesis of BCFA reduces lipoprotein-mediated TLR2 activation and improves lipase-mediated hydrolysis making it an important determinant of innate immunity. Overall, this study highlights the potential relevance of cell envelope acyl chain repertoire in infection dynamics of bacterial pathogens.     

Involvement of Toll-Like Receptors on Helicobacter pylori-Induced Immunity

Dendritic cells (DCs) play a major role in the innate immune response since they recognize a broad repertoire of PAMPs mainly via Toll-like receptors (TLRs). During Helicobacter pylori (H. pylori) infection, TLRs have been shown to be important to control cytokine response particularly in murine DCs. In the present study we analyzed the effect of blocking TLRs on human DCs. Co-incubation of human DCs with H. pylori resulted in the release of the pro-inflammatory cytokines IL-12p70, IL-6 and IL-10. Release of IL-12p70 and IL-10 was predominantly influenced when TLR4 signaling was blocked by adding specific antibodies, suggesting a strong influence on subsequent T cell responses through TLR4 activation on DCs. Co-incubation of H. pylori-primed DC with allogeneic CD4⁺ T cells resulted in the production of IFN-γ and IL-17A as well as the expression of Foxp3, validating a mixed Th1/Th17 and T_reg response in vitro. Neutralization of TLR4 during H. pylori infection resulted in significantly decreased amounts of IL-17A and IFN-γ and reduced levels of Foxp3-expressing and IL-10-secreting T cells. Our findings suggest that DC cytokine secretion induced upon TLR4-mediated recognition of H. pylori influences inflammatory and regulatory T cell responses, which might facilitate the chronic bacterial persistence.     

Opposing roles of IL-10 in acute bacterial infection

Interleukin-10 (IL-10) is recognized as an anti-inflammatory cytokine that downmodulates inflammatory immune responses at multiple levels. In innate cells, production of this cytokine is usually triggered after pathogen recognition receptor (PRR) engagement by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patters (DAMPs), as well as by other soluble factors. Importantly, IL-10 is frequently secreted during acute bacterial infections and has been described to play a key role in infection resolution, although its effects can significantly vary depending on the infecting bacterium. While the production of IL-10 might favor host survival in some cases, it may also result harmful for the host in other circumstances, as it can prevent appropriate bacterial clearance. In this review we discuss the role of IL-10 in bacterial clearance and propose that this cytokine is required to recover from infection caused by extracellular or highly pro-inflammatory bacteria. Altogether, we propose that IL-10 drives excessive suppression of the immune response upon infection with intracellular bacteria or in non-inflammatory bacterial infections, which ultimately favors bacterial persistence and dissemination within the host. Thus, the nature of the bacterium causing infection is an important factor that needs to be taken into account when considering new immunotherapies that consist on the modulation of inflammation, such as IL-10. Indeed, induction of this cytokine may significantly improve the host’s immune response to certain bacteria when antibiotics are not completely effective.     

Active Immunization with Extracellular Vesicles Derived from Staphylococcus aureus Effectively Protects against Staphylococcal Lung Infections,Mainly via Th1 Cell-Mediated Immunity

Staphylococcus aureus is an important pathogenic bacterium that causes various infectious diseases. Extracellular vesicles (EVs) released from S. aureus contain bacterial proteins, nucleic acids, and lipids. These EVs can induce immune responses leading to similar symptoms as during staphylococcal infection condition and have the potential as vaccination agent. Here, we show that active immunization (vaccination) with S. aureus-derived EVs induce adaptive immunity of antibody and T cell responses. In addition, these EVs have the vaccine adjuvant ability to induce protective immunity such as the up-regulation of co-stimulatory molecules and the expression of T cell polarizing cytokines in antigen-presenting cells. Moreover, vaccination with S. aureus EVs conferred protection against lethality induced by airway challenge with lethal dose of S. aureus and also pneumonia induced by the administration of sub-lethal dose of S. aureus. These protective effects were also found in mice that were adoptively transferred with splenic T cells isolated from S. aureus EV-immunized mice, but not in serum transferred mice. Furthermore, this protective effect of S. aureus EVs was significantly reduced by the absence of interferon-gamma, but not by the absence of interleukin-17. Together, the study herein suggests that S. aureus EVs are a novel vaccine candidate against S. aureus infections, mainly via Th1 cellular response.     

Timed Action of IL-27 Protects from Immunopathology while Preserving Defense in Influenza

Infection with influenza virus can result in massive pulmonary infiltration and potentially fatal immunopathology. Understanding the endogenous mechanisms that control immunopathology could provide a key to novel adjunct therapies for this disease. Here we show that the cytokine IL-27 plays a crucial role in protection from exaggerated inflammation during influenza virus infection. Using Il-27ra ^−/− mice, IL-27 was found to limit immunopathology, neutrophil accumulation, and dampened T_H1 or T_H17 responses via IL-10–dependent and -independent pathways. Accordingly, the absence of IL-27 signals resulted in a more severe disease course and in diminished survival without impacting viral loads. Consistent with the delayed expression of endogenous Il-27p28 during influenza, systemic treatment with recombinant IL-27 starting at the peak of virus load resulted in a major amelioration of lung pathology, strongly reduced leukocyte infiltration and improved survival without affecting viral clearance. In contrast, early application of IL-27 impaired virus clearance and worsened disease. These findings demonstrate the importance of IL-27 for the physiological control of immunopathology and the potential value of well-timed IL-27 application to treat life-threatening inflammation during lung infection.     

The incidence of Streptococcus pneumoniae otitis media is affected by the polymicrobial environment particularly Moraxella catarrhalis in a mouse nasal colonisation model

Otitis media (OM) is a highly prevalent paediatric disease with both bacterial and viral triggers of infection. This study has investigated how combinations of bacteria associated with nasal colonisation and the occurrence and absence of viral infection (Sendai virus) induce OM in a mouse nasal colonisation model. The respiratory virus significantly contributed to bacterial OM for all bacterial combinations (p < 0.001). Streptococcus pneumoniae consistently dominated as the causative bacterium of OM and when co-infected with S. pneumoniae, Moraxella catarrhalis more significantly affected pneumococcal OM than did non-typeable Haemophilus influenzae (p < 0.001) by increasing the incidence rate, infection bacterial load and duration of infection. Nitric oxide levels in the middle ear, an indicator of inflammation, peaked at day 3 in single bacterium groups, but at day 1 in mixed bacterial groups and was produced in all bacteria inoculated groups even in the absence of viable bacterial recovery. Phagocytic cells were recruited rapidly to the ear following nasal inoculation but over time their numbers did not correlate with persistence of bacterial infection. The study has shown that the composition of bacteria in the nasal cavity and respiratory viral infection significantly affected the OM incidence rate, duration of infection and bacterial load (severity).     

Elucidating the Crucial Role of Poly N-Acetylglucosamine from Staphylococcus aureus in Cellular Adhesion and Pathogenesis

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.