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1.  Proteomic Profiling of the Amniotic Fluid to Detect Inflammation, Infection, and Neonatal Sepsis 
PLoS Medicine  2007;4(1):e18.
Background
Proteomic analysis of amniotic fluid shows the presence of biomarkers characteristic of intrauterine inflammation. We sought to validate prospectively the clinical utility of one such proteomic profile, the Mass Restricted (MR) score.
Methods and Findings
We enrolled 169 consecutive women with singleton pregnancies admitted with preterm labor or preterm premature rupture of membranes. All women had a clinically indicated amniocentesis to rule out intra-amniotic infection. A proteomic fingerprint (MR score) was generated from fresh samples of amniotic fluid using surface-enhanced laser desorption ionization (SELDI) mass spectrometry. Presence or absence of the biomarkers of the MR score was interpreted in relationship to the amniocentesis-to-delivery interval, placental inflammation, and early-onset neonatal sepsis for all neonates admitted to the Newborn Special Care Unit (n = 104). Women with “severe” amniotic fluid inflammation (MR score of 3 or 4) had shorter amniocentesis-to-delivery intervals than women with “no” (MR score of 0) inflammation or even “minimal” (MR score of 1 or 2) inflammation (median [range] MR 3–4: 0.4 d [0.0–49.6 d] versus MR 1–2: 3.8 d [0.0–151.2 d] versus MR 0: 17.0 d [0.1–94.3 d], p < 0.001). Nonetheless, a “minimal” degree of inflammation was also associated with preterm birth regardless of membrane status. There was a significant association between the MR score and severity of histological chorioamnionitis (r = 0.599, p < 0.001). Furthermore, neonatal hematological indices and early-onset sepsis significantly correlated with the MR score even after adjusting for gestational age at birth (OR for MR 3–4: 3.3 [95% CI, 1.1 to 9.2], p = 0.03). When compared with other laboratory tests routinely used to diagnose amniotic fluid inflammation and infection, the MR score had the highest accuracy to detect inflammation (white blood cell count > 100 cells/mm3), whereas the combination of Gram stain and MR score was best for rapid prediction of intra-amniotic infection (positive amniotic fluid culture).
Conclusions
High MR scores are associated with preterm delivery, histological chorioamnionitis, and early-onset neonatal sepsis. In this study, proteomic analysis of amniotic fluid was shown to be the most accurate test for diagnosis of intra-amniotic inflammation, whereas addition of the MR score to the Gram stain provides the best combination of tests to rapidly predict infection.
Proteomic analysis of amniotic fluid in addition to a Gram stain provides the best combination of tests to rapidly predict intrauterine infection.
Editors' Summary
Background.
A preterm delivery, or premature birth, is normally defined as one that occurs before 37 weeks after the last menstrual cycle (an average pregnancy lasts around 40 weeks). Premature birth is fairly common, with around 12% of births in the US fitting this definition. However, it has serious consequences, being responsible for around 70% of infant deaths and other adverse outcomes for the baby. It is not clear in all cases what directly causes premature birth or how to identify cases in which mother and child are at greater risk of serious outcomes. Evidence from case-control and other studies strongly suggests that infections of the uterus, placenta, or genital tract are associated with, and are likely to directly cause, premature deliveries. Such infections, even if they are “subclinical” (that is, they do not directly cause signs or symptoms that the doctor or patient would notice) cause inflammation in the affected tissues. Hence, it's possible that particular proteins or other molecules could provide a “signature” that would allow the inflammation to be picked up at an early stage.
Why Was This Study Done?
If inflammation could be picked up early, this might help identify mothers at risk of having a preterm delivery, and even to pinpoint cases of very severe inflammation where the baby is more at risk of poor outcomes. The researchers involved in this study had already done previous work looking at protein profiles in the amniotic fluid (the liquid directly surrounding the developing fetus). They identified a set of four protein “markers” that were closely associated with inflammation in the amniotic fluid, and developed a score based on those proteins, which they termed the “Mass Restricted” (MR) score. The researchers showed that this score could accurately identify women at risk of preterm delivery. However, before using the protein marker score in clinical practice it is very important to really be sure it is a reliable diagnostic test for preterm birth and adverse outcomes resulting from preterm birth. Therefore the researchers wanted to find out whether MR scores were associated with the outcome of pregnancy; the presence of infection in the placenta, as detected through microscopic analysis of tissue; and sepsis (severe infection) in the newborn baby.
What Did the Researchers Do and Find?
The study was based on findings from pregnant women presenting at the Yale-New Haven Hospital with symptoms of premature labor, who were all followed up to the point of delivery of the baby. In all cases the decisions about how to manage the pregnancy (for example, whether to deliver the baby or attempt to delay birth) were made by the woman and her physician, not by any procedures laid out in the research study. A total of 169 women were recruited into the study and had a sample of amniotic fluid taken as part of their routine clinical management. The researchers then analyzed this fluid to calculate the protein MR score, to look for evidence of bacterial infection, and also carried out standard laboratory tests. After childbirth the placenta was examined under the microscope to look for any evidence of inflammation. Finally, all babies were checked for any evidence of sepsis. The researchers found that, in line with findings from their previous studies, women with a higher MR score gave birth sooner. There also seemed to be a close agreement between the MR score and evidence of inflammation in the placenta, once it was analyzed under the microscope after birth. Furthermore, mothers with a high MR score were more likely to give birth to babies with suspected or confirmed sepsis. The researchers then compared the usefulness of the MR score against other potential tests for inflammation. Of all the tests compared, the MR score seemed to be the most accurate in predicting inflammation.
What Do These Findings Mean?
This study showed that the MR score was closely associated with a number of different indicators of poor outcome in preterm birth. These outcomes included sooner deliveries, sepsis in the baby, and inflammation in the placenta. In future, the MR score may provide a useful test for recognizing women at risk of preterm delivery and babies at risk of poor outcome. However, further evaluation of the test will still need to be done before it could become a standard procedure in the clinic.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040018.
Information from the US National Institutes of Health on premature babies
The March of Dimes is a US charity that funds research into prematurity
Information from Wikipedia about proteomics the area of research used to develop the protein score examined here (note: Wikipedia is an online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040018
PMCID: PMC1769412  PMID: 17227133
2.  Ability of Procalcitonin to Discriminate Infection from Non-Infective Inflammation Using Two Pleural Disease Settings 
PLoS ONE  2012;7(12):e49894.
Procalcitonin has been shown to be useful in separating infection from non-infective disorders. However, infection is often paralleled by tissue inflammation. Most studies supporting the use of procalcitonin were confounded by more significant inflammation in the infection group. Few studies have examined the usefulness of procalcitonin when adjusted for inflammation.
Pleural inflammation underlies the development of most exudative effusions including pleural infection and malignancy. Pleurodesis, often used to treat effusions, involves provocation of intense aseptic pleural inflammation. We conducted a two-part proof-of-concept study to test the specificity of procalcitonin in differentiating infection using cohorts of patients with pleural effusions of infective and non-infective etiologies, as well as subjects undergoing pleurodesis.
Methods
We measured the blood procalcitonin level (i) in 248 patients with pleural infection or with non-infective pleural inflammation, matched for severity of systemic inflammation by C-reactive protein (CRP), age and gender; and (ii) in patients before and 24–48 hours after induction of non-infective pleural inflammation (from talc pleurodesis).
Results
1) Procalcitonin was significantly higher in patients with pleural infection compared with controls with non-infective effusions (n = 32 each group) that were case-matched for systemic inflammation as measured by CRP [median (25–75%IQR): 0.58 (0.35–1.50) vs 0.34 (0.31–0.42) µg/L respectively, p = 0.003]. 2) Talc pleurodesis provoked intense systemic inflammation, and raised serum CRP by 360% over baseline. However procalcitonin remained relatively unaffected (21% rise). 3) Procalcitonin and CRP levels did not correlate. In 214 patients with pleural infection, procalcitonin levels did not predict the survival or need for surgical intervention.
Conclusion
Using a pleural model, this proof-of-principle study confirmed that procalcitonin is a biomarker specific for infection and is not affected by non-infective inflammation. Procalcitonin is superior to CRP in distinguishing infection from non-infective pleural diseases, even when controlled for the level of systemic inflammation.
doi:10.1371/journal.pone.0049894
PMCID: PMC3520973  PMID: 23251353
3.  Multiple Chlamydiaceae Species in Trachoma: Implications for Disease Pathogenesis and Control 
PLoS Medicine  2008;5(1):e14.
Background
Chlamydia trachomatis is a unique obligate intracellular bacterium that remains the leading cause of sexually transmitted bacterial diseases and preventable blindness worldwide. Chronic ocular infections are referred to as trachoma, and predominate in developing countries. Since 2001, the World Health Organization has promoted control strategies including antibiotics, improved hygiene, and environmental measures with limited success. Consequently, a vaccine is urgently needed. Integral to vaccine design is an understanding of the interactions of the pathogen and host immune response. Various animal models of trachoma show that urogenital C. trachomatis strains and other species of the family Chlamydiaceae produce severe conjunctival inflammation and scarring similar to that of the ocular C. trachomatis strains. However, we do not know the extent of organisms that may be involved in human trachoma. Furthermore, C. trachomatis heat shock protein 60 (Hsp60) has been implicated in inflammation and conjunctival scarring but the role of other Chlamydiaceae Hsp60 in disease pathogenesis has not been examined. In this study, we set out to identify whether other Chlamydiaceae species are present in trachoma, and determine their association with severity of clinical disease and with mucosal and systemic immune responses to Chlamydiaceae species-specific Hsp60 to further investigate the immunopathogenesis of this blinding disease.
Methods and Findings
We randomly selected nine of 49 households in a trachoma-endemic region of Nepal. Trachoma was graded, and real-time, quantitative (k)PCR was used to detect genomic DNA and cDNA (from RNA) for Chlamydiaceae ompA and 16S rRNA genes, respectively, from conjunctival swabs. IgG antibody responses to recombinant (r) Chlamydiaceae species-specific Hsp60 were determined for tears and sera. Surprisingly, all three species—C. trachomatis, Chlamydophila psittaci, and Chlamydophila pneumoniae—were detected in eight (89%) study households; one household had no members infected with C. pneumoniae. Of 80 (63%; n = 127) infected individuals, 28 (35%) had infection with C. psittaci, or C. pneumoniae, or both; single and dual infections with C. psittaci and C. pneumoniae were significantly associated with severe conjunctival inflammation (OR 4.25 [95% confidence interval (CI), 2.9–11.3], p = 0.009] as were single infections with C. trachomatis (OR 5.7 [95% CI, 3.8–10.1], p = 0.002). Of the 80 infected individuals, 75 (93.8%) were also positive for 16S rRNA by kPCR for the same organism identified by ompA. Individuals with tear IgG immunoreactivity to Chlamydiaceae rHsp60 were eight times more likely than individuals without tear immunoreactivity to be infected (95% CI 6.4–15.1; p = 0.003), 6.2 times more likely to have severe inflammation (95% CI 4.4–12.6; p = 0.001), and 5.7 times more likely to have scarring (95% CI 3.9–11.1; p = 0.019) while individuals with serum IgG immunoreactivity were 4.1 times more likely to be infected (95% CI 3.1–10.1; p = 0.014).
Conclusions
We provide substantial evidence for the involvement of C. psittaci and C. pneumoniae, in addition to C. trachomatis, in trachoma. The distribution of Chlamydiaceae species by household and age suggests that these infections are widespread and not just sporadic occurrences. Infection with multiple species may explain the failure to detect chlamydiae among active trachoma cases, when only C. trachomatis is assayed for, and the failure of clinically active cases to resolve their disease following what would be considered effective C. trachomatis treatment. The evidence for viable (RNA-positive) organisms of all three species in single and coinfections, the significant association of these infections with severe inflammation, and the significant association of tear and serum IgG responses to Chlamydiaceae Hsp60 with inflammation and scarring, support the role of all three species in disease pathogenesis. Thus, while our findings should be confirmed in other trachoma-endemic countries, our data suggest that a reevaluation of treatment regimens and vaccine design may be required. Understanding the full impact of Chlamydiaceae species on the epidemiology, immunopathology, and disease outcome of trachoma presents a new challenge for Chlamydiaceae research.
In a study of trachoma cases within households in Nepal, Deborah Dean and colleagues find involvement of the Chlamydia species C. psittaci and C. pneumoniae in addition to C. trachomatis.
Editors' Summary
Background.
Six million people—most of whom live in crowded, unhygienic conditions in developing countries—are blind because of an infectious disease called trachoma. It is generally accepted that trachoma is caused by Chlamydia trachomatis, bacteria that pass easily between people on hands and clothing. Infection usually occurs first during childhood, but people do not become blind until adulthood. Successive infections cause progressive scarring of the inside of the eyelid. Eventually, the eyelashes turn inward and rub painfully over the front of the eye (the cornea). This causes corneal scarring, loss of corneal transparency and, finally, irreversible blindness. C. trachomatis infections can be prevented by improving personal hygiene (in particular, facial cleanliness in children) and by reducing fly breeding sites, and they can be treated with antibiotics. However, C. trachomatis and other organisms appear to be developing drug resistance to antibiotics commonly used to treat these infections. In addition, early scarring and in-turned eyelashes can be treated surgically, although recurrence of the in-turned eyelashes frequently occurs months to years after surgery.
Why Was This Study Done?
The World Health Organization has been promoting these “SAFE” interventions (surgery, antibiotics, facial cleanliness, and environmental improvement) since 2001 with the aim of eliminating trachoma by 2020. However, these control measures have had limited success so far and it looks like a vaccine may also be needed. To develop an effective vaccine, scientists need to know whether all cases of human trachoma are caused by so-called ocular strains of C. trachomatis. Might C. trachomatis strains that are usually associated with sexually transmitted disease (urogenital strains) or different species in the family Chlamydiaceae also cause human trachoma as work in animals has suggested? In this study, the researchers have investigated which Chlamydiaceae species are associated with trachoma in a region of Nepal where the disease is endemic (always present).
What Did the Researchers Do and Find?
The researchers examined all the members for trachoma in nine randomly selected households in a Nepali village. They then used sensitive molecular biology methods to identify the species in the family Chlamydiaceae and strains present in the eyes of the infected individuals. One third of them were infected with only C. trachomatis (mainly ocular strains but also some urogenital strains), one in five were infected with only Chlamydophila psittaci, and one in ten with only Chlamydophila pneumoniae. The other infected individuals had mixed infections. Infection with C. psittaci and/or C. pneumoniae was strongly associated with severe eye inflammation as was infection with C. trachomatis alone. The researchers also asked whether there were any antibodies (proteins made by the immune system that recognize infectious organisms) in the tears or blood of the infected individuals that recognized the Hsp60 protein of each Chlamydiaceae species; an immune response to C. trachomatis Hsp60 is thought to be involved in the inflammation and scarring seen in trachoma. Individuals with antibodies in their tears to Chlamydiaceae Hsp60, the researchers report, were eight times as likely to be actively infected with these bacteria and six times as likely to have severe eye inflammation as individuals without the antibodies.
What Do These Findings Mean?
These findings provide evidence for the widespread involvement of C. psittaci, C. pneumoniae, and urogenital strains of C. trachomatis as well as ocular strains of C. trachomatis in trachoma and might explain why some people with active trachoma do not have C. trachomatis in their eye secretions and why antibiotics that kill C. trachomatis effectively do not cure all cases of trachoma. However, because live bacteria were not isolated from patients and shown to cause disease in a model system, these findings do not prove that Chlamydiaceae other than C. trachomatis cause trachoma. Some or all of the bacterial strains and species detected in this study may be innocent bystanders although the strong association between their presence and severe inflammation and the association between antibody responses to Chlamydiaceae Hsp60 and inflammation argues against this possibility. If the involvement of multiple Chlamydiaceae strains and species is confirmed and extended in other trachoma-endemic regions, then future antimicrobial therapies and vaccines will need to deal with all these bacteria and not just C. trachomatis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050014.
The MedlinePlus encyclopedia contains a page on trachoma (in English and Spanish)
The World Health Organization provides information on trachoma (mainly in English but some information is available in French, Russian, and Spanish)
The US Centers for Disease Control and Prevention provides a technical fact sheet on trachoma
The charity Sightsavers International also provides information on trachoma and global efforts to eliminate the disease
The Carter Center provides an overview of trachoma control and a description of its trachoma control program
doi:10.1371/journal.pmed.0050014
PMCID: PMC2174965  PMID: 18177205
4.  Haemophilus influenzae Infection Drives IL-17-Mediated Neutrophilic Allergic Airways Disease 
PLoS Pathogens  2011;7(10):e1002244.
A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12–15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.
Author Summary
Approximately 50% of asthmatics have non-eosinophilic inflammation, and 20% of these patients have severe neutrophilic inflammation and increased IL-8 levels. These so-called neutrophilic asthmatics have persistent airway colonization with bacteria, and Haemophilus influenzae is one of the bacteria most commonly isolated. However, how H. influenzae is associated with the pathogenesis of neutrophilic asthma is unknown. In this study we used mouse models to investigate the relationship between H. influenzae infection and allergic airways disease (AAD). We showed that infection promoted the development of hallmark features of neutrophilic asthma. Infection suppressed Th2 cytokines, eosinophilic inflammation, and AHR in AAD, while increasing neutrophilic inflammation and IL-17 responses. Importantly, inhibition of IL-17 during AAD reduced airway neutrophils and neutrophil chemokines, suggesting that infection drives the development of neutrophilic inflammation through an IL-17-mediated mechanism. This provides novel insights into the mechanisms that may underpin infection-induced neutrophilic asthma. These data also suggest that treatments targeting infection may lead to improved management of neutrophilic asthma.
doi:10.1371/journal.ppat.1002244
PMCID: PMC3188527  PMID: 21998577
5.  Parasitic Nematode-Induced CD4+Foxp3+T Cells Can Ameliorate Allergic Airway Inflammation 
Background
The recruitment of CD4+CD25+Foxp3+T (Treg) cells is one of the most important mechanisms by which parasites down-regulate the immune system.
Methodology/Principal Findings
We compared the effects of Treg cells from Trichinella spiralis-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced Treg cells. After four weeks of T. spiralis infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4+Foxp3+ cells from T. spiralis-infected [Inf(+)Foxp3+] or uninfected [Inf(-)Foxp3+] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3+ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3+ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3+ cells migrated to inflammation sites in the lung and expressed higher levels of Treg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3+ cells.
Conclusion/Significance
T. spiralis infection promotes the proliferation and functional activation of Treg cells. Parasite-induced Treg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced Treg cells. The adoptive transfer of Inf(+)Foxp3+ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment of allergic airway diseases.
Author Summary
Many studies have investigated the down-regulation of the immune system by parasite infection. CD4+CD25+Foxp3+T (Treg) cells are key players in parasite-mediated immune downregulation. Our previous study suggested that Treg cells recruited by Trichinella spiralis infection were the key cells mediating the amelioration of allergic airway inflammation in mice. In the present study, we investigated the functions of parasite-induced Treg cells using mice expressing GFP-tagged Foxp3. T. spiralis infection increased the number of Treg cells. Adoptive transfer of the parasite-induced Treg cells to mice with allergic airway inflammation ameliorated allergic airway inflammation. The transferred cells were recruited to inflammation sites in the lung. Cells from parasite-infected mice expressed higher levels of Treg-cell homing receptors and activation markers than did cells from uninfected mice. This study might help explain why immune disorders (often of unknown cause) are more prevalent among people in developed countries (areas with low parasite infection) than among those in developing countries (areas with parasite epidemics). Our finding might improve current cell therapy techniques and facilitate the development of new techniques that use parasites or parasite-borne materials to treat diverse immune disorders.
doi:10.1371/journal.pntd.0003410
PMCID: PMC4270642  PMID: 25522145
6.  Distinct Lipid A Moieties Contribute to Pathogen-Induced Site-Specific Vascular Inflammation 
PLoS Pathogens  2014;10(7):e1004215.
Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE−/− mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE−/− mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.
Author Summary
Several human pathogens express structurally divergent forms of lipid A, the endotoxic portion of lipopolysaccharide (LPS), as a strategy to evade host innate immune detection and establish persistent infection. Expression of modified lipid A species promotes pathogen evasion of host recognition by Toll-like receptor-4 (TLR4) and the non-canonical inflammasome. The Gram-negative oral anaerobe, Porphyromonas gingivalis, expresses lipid A structures that function as TLR4 agonists or antagonists, or are immunologically inert. It is currently unclear how modulation of P. gingivalis lipid A expression contributes to innate immune recognition, survival, and the ability of the pathogen to induce local and systemic inflammation. In this study, we demonstrate that P. gingivalis expression of antagonist lipid A species results in attenuated production of proinflammatory mediators and evasion of non-canonical inflammasome activation, facilitating bacterial survival in the macrophage. Infection of atherosclerosis-prone ApoE−/− mice with this strain resulted in progression of chronic inflammation in the vasculature. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A modifications, supporting distinct mechanisms for induction of local versus systemic inflammation. Our work demonstrates that evasion of immune detection at TLR4 contributes to pathogen persistence and facilitates low-grade chronic inflammation.
doi:10.1371/journal.ppat.1004215
PMCID: PMC4092147  PMID: 25010102
7.  IL-37 Inhibits Inflammasome Activation and Disease Severity in Murine Aspergillosis 
PLoS Pathogens  2014;10(11):e1004462.
Since IL-37 transgenic mice possesses broad anti-inflammatory properties, we assessed whether recombinant IL-37 affects inflammation in a murine model of invasive pulmonary aspergillosis. Recombinant human IL-37 was injected intraperitoneally into mice prior to infection and the effects on lung inflammation and inflammasome activation were evaluated. IL-37 markedly reduced NLRP3-dependent neutrophil recruitment and steady state mRNA levels of IL-1β production and mitigated lung inflammation and damage in a relevant clinical model, namely aspergillosis in mice with cystic fibrosis. The anti-inflammatory activity of IL-37 requires the IL-1 family decoy receptor TIR-8/SIGIRR. Thus, by preventing activation of the NLRP3 inflammasome and reducing IL-1β secretion, IL-37 functions as a broad spectrum inhibitor of the innate response to infection-mediated inflammation, and could be considered to be therapeutic in reducing the pulmonary damage due to non-resolving Aspergillus infection and disease.
Author Summary
IL-37, firstly identified by in silico research in the year 2000, is a member of the IL-1 family. The biological properties of IL-37 are mainly those of down-regulating inflammation in models of septic shock, chemical colitis, cardiac ischemia and contact dermatitis. Whether and how IL-37 down-regulates the inflammation of infection, and its consequences, is not known. We observed that IL-37 limits inflammation and disease severity in murine invasive aspergillosis, an infection model in which cytokines of the IL-1 family have important roles. However, given that IL-1R1-deficient or caspase 1-deficient mice are resistant to lung inflammation during infection and that IL-1 signaling could drive the differentiation of antifungal inflammatory Th17 cells, the pro-inflammatory properties of IL 1-induced inflammation in aspergillosis is potentially dangerous for the host. IL-37 markedly reduced NLRP3-dependent neutrophil recruitment and steady state mRNA levels of IL-1β production and mitigated lung inflammation and damage in a relevant clinical model, namely aspergillosis in mice with cystic fibrosis. The anti-inflammatory activity of IL-37 requires the IL-1 receptor family decoy TIR-8/SIGIRR. Thus, IL-37 functions as a broad spectrum inhibitor of infection-mediated inflammation, and could be considered to be therapeutic in reducing the pulmonary damage due to non-resolving Aspergillus infection and disease.
doi:10.1371/journal.ppat.1004462
PMCID: PMC4223056  PMID: 25375146
8.  IL-1β Production through the NLRP3 Inflammasome by Hepatic Macrophages Links Hepatitis C Virus Infection with Liver Inflammation and Disease 
PLoS Pathogens  2013;9(4):e1003330.
Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.
Author Summary
Hepatitis C virus (HCV) causes chronic infection of the liver and is a leading cause of liver inflammation, cirrhosis and liver cancer in nearly 200 million people worldwide. Importantly, hepatic inflammation during chronic HCV infection is considered to be the primary catalyst for progressive liver disease and development of liver cancer. However, the underlying molecular mechanism(s) of HCV-mediated hepatic inflammation are not well understood. The goal of this study was to determine the mechanisms of HCV-induced inflammation. We found that serum IL-1β levels are elevated in chronic hepatitis C patients. Furthermore, we found that hepatic macrophages or Kupffer cells are the major IL-1β-producing cell population within HCV infected livers. Our studies, using the THP1 cell culture model of HCV exposure, reveal that exposure of macrophages to HCV induces IL-1β through a process of infection-independent phagocytic virus uptake that triggers signaling through MyD88/TLR7 and NLRP3 inflammasome pathways to drive IL-1β expression and maturation/secretion, respectively. RNA sequencing (RNA-seq) analysis of patient liver biopsies shows that viral triggering of these signaling pathways drives an inflammatory response linked with liver disease in patients with chronic hepatitis C. Our results identify HCV-induced IL-1β production by hepatic macrophages as a critical and central process that promotes liver inflammation and disease.
doi:10.1371/journal.ppat.1003330
PMCID: PMC3635973  PMID: 23633957
9.  Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection 
Background
Worldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences.
Methods
To better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5–12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or αPD1 ligand were studied.
Results
Twelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation.
Conclusion
In outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.
doi:10.1186/1742-2094-5-48
PMCID: PMC2588578  PMID: 18947414
10.  Extent of liver inflammation in predicting response to interferon α & Ribavirin in chronic hepatitis C patients: a cohort study 
BMC Gastroenterology  2012;12:71.
Background
Liver inflammation due to HCV infection leads to fibrosis, which is an independent predictor of treatment response to interferon therapy in Chronic Hepatitis C (CHC) patients. This relationship has not been studied for liver inflammation on pretreatment liver biopsy and End of Treatment Response (ETR). ALT is a less invasive test than liver biopsy for measuring liver inflammation. Aim of this study was to compare ETR to Interferon α (recombinant Interferon) & Ribavirin in CHC patients having higher and lower grades of liver inflammation and to determine the diagnostic accuracy of pretreatment ALT for grades of liver inflammation.
Methods
A retrospective cohort of 876 naïve CHC patients, who completed Interferon α & Ribavirin for 24 weeks, was studied for ETR. Pretreatment grade of inflammation on liver biopsy was taken as the exposure variable. It was classified as high if there was moderate or severe and low if there was minimal or mild. Multivariable logistic regression modeling was performed. Diagnostic accuracy of pretreatment ALT for liver inflammation grades was determined by computing Area Under the Receiver Operator Curve (AUROC).
Results
Of all patients, 672 having diagnostic liver biopsy and ETR available were analyzed. Among them, 103 had high and 569 had low grades of liver inflammation. Mean age was 36.9 (SD 9.1) years, with patients with high grades being older than those with low grades inflammation (p = 0.03). High grades of liver inflammation was associated with ETR (RR 1.17, 95% CI 1.12–1.18) adjusting for age, Total Leukocyte count (TLC) and pretreatment levels of ALT, irrespective of liver fibrosis. This relation remained significant for ‘bridging fibrosis and cirrhosis’ and not for ‘no’ or ‘portal fibrosis’. AUROC of pretreatment ALT for males and females was moderately accurate for severe inflammation compared to minimal inflammation and less accurate for high grades compared to low grades.
Conclusions
ETR in patients with higher grades of liver inflammation was 17% higher than those with lower grades irrespective of fibrosis and 9% higher for bridging fibrosis and cirrhosis. Pretreatment ALT was moderately accurate for severe inflammation only on liver biopsy in both males and females.
doi:10.1186/1471-230X-12-71
PMCID: PMC3502580  PMID: 22697612
11.  Foreskin inflammation is associated with HIV and herpes simplex virus type-2 infections in Rakai, Uganda 
AIDS (London, England)  2009;23(14):1807-1815.
Design
We assessed foreskin inflammation associated with HIV and herpes simplex virus type 2 (HSV-2) in circumcised men.
Methods
Foreskin tissues were assessed in 97 HIV-infected and 135 HIV-uninfected men enrolled in randomized trials of circumcision in Rakai, Uganda. Inflammation was quantified using an ordinal score evaluating extent, intensity, and cellular composition of infiltrates in the epithelium and stroma. Prevalence rate ratios of inflammation were estimated by multivariate Poisson regression.
Results
Foreskin inflammation was primarily focal. Epithelial inflammation was present in 4.2% of men with neither HIV nor HSV-2 infection; 7.8% of men with only HSV-2; 19.0% with HIV alone (P=0.04); and 31.6% in HIV/HSV-2 coinfected men [prevalence rate ratio (PRR) 7.5, 95% confidence interval (CI) 2.3-23.8, P<0.001]. Stromal inflammation was present in 14.1% of HIV/HSV-2 uninfected men, compared with 29.7% in men with HSV-2 alone (P=0.03), 33.3% in men with HIV alone (P=0.04), and 61.0% in men with HIV/HSV-2 coinfection (PRR 4.3, 95% CI 2.3-7.9, P<0.001). In HIV-infected men, epithelial inflammation was associated with higher HIV viral load. Epithelial inflammation was more frequent among men reporting recent genital ulceration. Both epithelial and stromal inflammation were more common among men with smegma on physical examination.
Conclusion
Foreskin inflammation is increased with HIV and HSV-2 infections, higher HIV viral load and presence of smegma. Foreskin inflammation may have implications for HIV transmission and acquisition in uncircumcised men.
doi:10.1097/QAD.0b013e32832efdf1
PMCID: PMC2752438  PMID: 19584700
circumcision; foreskin; HIV; herpes simplex virus type 2; inflammation; Uganda
12.  Granulocytes Impose a Tight Bottleneck upon the Gut Luminal Pathogen Population during Salmonella Typhimurium Colitis 
PLoS Pathogens  2014;10(12):e1004557.
Topological, chemical and immunological barriers are thought to limit infection by enteropathogenic bacteria. However, in many cases these barriers and their consequences for the infection process remain incompletely understood. Here, we employed a mouse model for Salmonella colitis and a mixed inoculum approach to identify barriers limiting the gut luminal pathogen population. Mice were infected via the oral route with wild type S. Typhimurium (S. Tm) and/or mixtures of phenotypically identical but differentially tagged S. Tm strains (“WITS”, wild-type isogenic tagged strains), which can be individually tracked by quantitative real-time PCR. WITS dilution experiments identified a substantial loss in tag/genetic diversity within the gut luminal S. Tm population by days 2–4 post infection. The diversity-loss was not attributable to overgrowth by S. Tm mutants, but required inflammation, Gr-1+ cells (mainly neutrophilic granulocytes) and most likely NADPH-oxidase-mediated defense, but not iNOS. Mathematical modelling indicated that inflammation inflicts a bottleneck transiently restricting the gut luminal S. Tm population to approximately 6000 cells and plating experiments verified a transient, inflammation- and Gr-1+ cell-dependent dip in the gut luminal S. Tm population at day 2 post infection. We conclude that granulocytes, an important clinical hallmark of S. Tm-induced inflammation, impose a drastic bottleneck upon the pathogen population. This extends the current view of inflammation-fuelled gut-luminal Salmonella growth by establishing the host response in the intestinal lumen as a double-edged sword, fostering and diminishing colonization in a dynamic equilibrium. Our work identifies a potent immune defense against gut infection and reveals a potential Achilles' heel of the infection process which might be targeted for therapy.
Author Summary
Salmonella Typhimurium can colonize the human intestine and cause severe diarrhea. In recent years, it has become clear that this pathogen profits from inflammatory changes in the intestinal lumen, as the inflamed gut helps Salmonella to out-compete the resident microbiota. Granulocytes transmigrating into the gut lumen were found to “foster” luminal Salmonella growth by providing nutrients (used by Salmonella, not the microbiota) and by releasing growth inhibitors affecting the microbiota, but not the pathogen. In this study, we extend this “fostering” concept by showing that gut luminal Salmonella Typhimurium population is itself surprisingly vulnerable to the host's inflammatory response. Indeed, inflammation reduces the size of the gut luminal Salmonella population by as much as 105-fold at day 2 post infection. Thus, triggering of mucosal inflammation is in fact a double-edged sword by providing S. Typhimurium with a relative growth advantage against the microbiota in the gut lumen and by killing 99.999% of the gut luminal pathogen population at day 2. However, the pathogen population can recover and grow up again during the subsequent days. This changes the current view: Inflammation is not simply “beneficial” for the pathogen in the gut lumen. Instead, pathogen growth in the inflamed gut must be considered as an equilibrium between inflammation-inflicted killing and fostering growth of the surviving bacteria.
doi:10.1371/journal.ppat.1004557
PMCID: PMC4270771  PMID: 25522364
13.  DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway 
PLoS Pathogens  2014;10(1):e1003848.
Pathogen-associated molecular patterns (PAMPs) trigger host immune response by activating pattern recognition receptors like toll-like receptors (TLRs). However, the mechanism whereby several pathogens, including viruses, activate TLRs via a non-PAMP mechanism is unclear. Endogenous “inflammatory mediators” called damage-associated molecular patterns (DAMPs) have been implicated in regulating immune response and inflammation. However, the role of DAMPs in inflammation/immunity during virus infection has not been studied. We have identified a DAMP molecule, S100A9 (also known as Calgranulin B or MRP-14), as an endogenous non-PAMP activator of TLR signaling during influenza A virus (IAV) infection. S100A9 was released from undamaged IAV-infected cells and extracellular S100A9 acted as a critical host-derived molecular pattern to regulate inflammatory response outcome and disease during infection by exaggerating pro-inflammatory response, cell-death and virus pathogenesis. Genetic studies showed that the DDX21-TRIF signaling pathway is required for S100A9 gene expression/production during infection. Furthermore, the inflammatory activity of extracellular S100A9 was mediated by activation of the TLR4-MyD88 pathway. Our studies have thus, underscored the role of a DAMP molecule (i.e. extracellular S100A9) in regulating virus-associated inflammation and uncovered a previously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in regulating inflammation during infection.
Author Summary
The lung disease severity following influenza A virus (IAV) infection is dependent on the extent of inflammation in the respiratory tract. Severe inflammation in the lung manifests in development of pneumonia. Therefore, it is very critical to identify cellular factors and dissect the molecular/cellular mechanism controlling inflammation in the respiratory tract during IAV infection. Knowledge derived from these studies will be instrumental in development of therapeutics to combat the lung disease associated with IAV infection. Towards that end, in the current study we have identified a cellular factor S100A9 which is responsible for enhanced inflammation during IAV infection. In addition, we have characterized a signal transduction pathway involving various cellular receptors and signaling adaptors that are involved in mediating S100A9-dependent inflammatory response. Thus, our studies have illuminated a cellular/molecular mechanism that can be intervened by therapeutics to reduce and control IAV-associated lung inflammatory disease like pneumonia.
doi:10.1371/journal.ppat.1003848
PMCID: PMC3879357  PMID: 24391503
14.  Late Engagement of CD86 after Influenza Virus Clearance Promotes Recovery in a FoxP3+ Regulatory T Cell Dependent Manner 
PLoS Pathogens  2014;10(8):e1004315.
Influenza A virus (IAV) infection in the respiratory tract triggers robust innate and adaptive immune responses, resulting in both virus clearance and lung inflammation and injury. After virus clearance, resolution of ongoing inflammation and tissue repair occur during a distinct recovery period. B7 family co-stimulatory molecules such as CD80 and CD86 have important roles in modulating T cell activity during the initiation and effector stages of the host response to IAV infection, but their potential role during recovery and resolution of inflammation is unknown. We found that antibody-mediated CD86 blockade in vivo after virus clearance led to a delay in recovery, characterized by increased numbers of lung neutrophils and inflammatory cytokines in airways and lung interstitium, but no change in conventional IAV-specific T cell responses. However, CD86 blockade led to decreased numbers of FoxP3+ regulatory T cells (Tregs), and adoptive transfer of Tregs into αCD86 treated mice rescued the effect of the blockade, supporting a role for Tregs in promoting recovery after virus clearance. Specific depletion of Tregs late after infection mimicked the CD86 blockade phenotype, confirming a role for Tregs during recovery after virus clearance. Furthermore, we identified neutrophils as a target of Treg suppression since neutrophil depletion in Treg-depleted mice reduced excess inflammatory cytokines in the airways. These results demonstrate that Tregs, in a CD86 dependent mechanism, contribute to the resolution of disease after IAV infection, in part by suppressing neutrophil-driven cytokine release into the airways.
Author Summary
Influenza A virus (IAV) infection can cause severe inflammation and injury in the respiratory tract, which must be resolved and repaired for the host to fully recover after virus clearance. Evidence is emerging that host immune responses may regulate tissue repair and resolution of inflammation after IAV infection. Early in IAV infection, the co-stimulatory molecules CD80 and CD86 promote inflammation through triggering IAV-specific T cell responses, but no role for CD80/86 in recovery after virus clearance has been previously established. By in vivo antibody-mediated blockade of CD80 or CD86 after virus clearance, we found that engagement of CD86 (but not CD80) was required for optimal recovery after influenza infection. Furthermore, we determined that CD86 was essential for maintaining the FoxP3+regulatory T cell (Treg) population in the respiratory tract, and CD86-dependent Tregs promoted recovery by suppressing pulmonary inflammation and supporting regain of weight after virus clearance. In addition, we demonstrated that Tregs suppress neutrophils late after infection, preventing neutrophils from driving excess inflammatory cytokine release into the airways. Taken together, we propose a novel role for CD86 engagement late after IAV infection to promote resolution of inflammation and host recovery through a Treg-dependent mechanism.
doi:10.1371/journal.ppat.1004315
PMCID: PMC4140856  PMID: 25144228
15.  Host Defense and Recruitment of Foxp3+ T Regulatory Cells to the Lungs in Chronic Mycobacterium tuberculosis Infection Requires Toll-like Receptor 2 
PLoS Pathogens  2013;9(6):e1003397.
Acute resistance to low dose M. tuberculosis (Mtb) infection is not dependent on Toll-like receptor (TLR) 2. However, whether TLR2 contributes to resistance in chronic Mtb infection has remained uncertain. Here we report that, following low dose aerosol infection with Mtb, mice lacking TLR2 (TLR2KO), in comparison with wild type (WT) mice, exhibit enhanced cellular infiltration and inflammation in the lungs, and fail to stably control bacterial burden during chronic infection. IFNγ and IL-17 was expressed at equivalent levels in the two groups; however, the characteristic accumulation of Foxp3+ T regulatory cells (Tregs) in pulmonary granulomas was significantly reduced in TLR2KO mice. Nonetheless, this reduction in Tregs was independent of whether Tregs expressed TLR2 or not. To directly link the reduced number of Tregs to the increased inflammation present in the TLR2KO mice, we used a macrophage adoptive transfer model. At seven weeks post-Mtb infection, TLR2KO mice, which were adoptively transferred with WT macrophages, displayed enhanced accumulation of Tregs in the lungs and a concomitant reduction in inflammation in contrast with control mice that received TLR2KO macrophages. However, the pulmonary bacterial burden between the two groups remained similar indicating that TLR2's role in modulating immunopathology is functionally distinct from its role in restricting Mtb growth in chronic infection. Together, these findings unequivocally demonstrate that TLR2 contributes to host resistance against chronic Mtb infection and reveal a novel role for TLR2 in mediating the recruitment of Foxp3+ Tregs to the lungs to control inflammation.
Author Summary
Tuberculosis (TB) is an important cause of mortality in many parts of the world. Infection with Mycobacterium tuberculosis (Mtb), the causative agent of TB, is usually acquired via inhalation of airborne droplets containing the bacteria. Following inhalation, Mtb interacts with specialized receptors, called Toll-like receptors (TLRs), on phagocytic cells present in the lung. In this study, we examine the contribution of TLR2 in activating the body's natural defenses against Mtb. Wild type mice infected with Mtb by the aerosol route are able to control bacterial replication in the lung and maintain it at a steady level during chronic infection. However, in genetically modified mice that do not express TLR2 (TLR2KO), Mtb infection leads to increased inflammation in the lung and inability to control Mtb growth. Here, we identify that the increased inflammation present in the lungs of Mtb-infected TLR2KO mice is due to the diminished ability of a type of regulatory cell (Foxp3+ Tregs) to accumulate in the lungs. The ability to recruit Tregs to the lungs is restored in TLR2KO mice if they are adoptively transferred with macrophages from wild type mice. In summary, we demonstrate that TLR2 functions in protection against chronic Mtb infection by controlling Treg accumulation in the lung to limit inflammation and tissue damage.
doi:10.1371/journal.ppat.1003397
PMCID: PMC3681744  PMID: 23785280
16.  The role of inflammation in HPV infection of the Oesophagus 
BMC Cancer  2013;13:185.
Background
Several human cancers are known to be associated with inflammation and/or viral infections. However, the influence of tumour-related inflammation on viral uptake is largely unknown. In this study we used oesophageal squamous cell carcinoma (OSCC) as a model system since this type of cancer is associated with chronic irritation, inflammation and viral infections. Although still debated, the most important viral infection seems to be with Human Papillomavirus (HPV). The present study focused on a possible correlation between inflammation, OSCC development and the influence of HPV infection.
Methods
A total of 114 OSCC biopsies and corresponding normal tissue were collected at Groote Schuur Hospital and Tygerberg Hospital, Cape Town (South Africa), that were subjected to RNA and DNA isolation. RNA samples were analysed by quantitative Light Cycler RT-PCR for the expression of selected genes involved in inflammation and infection, while conventional PCR was performed on the DNA samples to assess the presence of integrated viral DNA. Further, an in vitro infection assay using HPV pseudovirions was established to study the influence of inflammation on viral infectivity using selected cell lines.
Results
HPV DNA was found in about 9% of OSCC patients, comprising predominantly the oncogenic type HPV18. The inflammatory markers IL6 and IL8 as well as the potential HPV receptor ITGA6 were significantly elevated while IL12A was downregulated in the tumour tissues. However, none of these genes were expressed in a virus-dependent manner. When inflammation was mimicked with various inflammatory stimulants such as benzo-α-pyrene, lipopolysaccharide and peptidoglycan in oesophageal epithelial cell lines in vitro, HPV18 pseudovirion uptake was enhanced only in the benzo-α-pyrene treated cells. Interestingly, HPV pseudovirion infectivity was independent of the presence of the ITGA6 receptor on the surface of the tested cells.
Conclusion
This study showed that although the carcinogen benzo-α-pyrene facilitated HPV pseudovirion uptake into cells in culture, HPV infectivity was independent of inflammation and seems to play only a minor role in oesophageal cancer.
doi:10.1186/1471-2407-13-185
PMCID: PMC3623831  PMID: 23570247
HPV; Cytokines; Receptors; Oesophageal cancer
17.  Innate Immune Responses and Rapid Control of Inflammation in African Green Monkeys Treated or Not with Interferon-Alpha during Primary SIVagm Infection 
PLoS Pathogens  2014;10(7):e1004241.
Chronic immune activation (IA) is considered as the driving force of CD4+ T cell depletion and AIDS. Fundamental clues in the mechanisms that regulate IA could lie in natural hosts of SIV, such as African green monkeys (AGMs). Here we investigated the role of innate immune cells and IFN-α in the control of IA in AGMs. AGMs displayed significant NK cell activation upon SIVagm infection, which was correlated with the levels of IFN-α. Moreover, we detected cytotoxic NK cells in lymph nodes during the early acute phase of SIVagm infection. Both plasmacytoid and myeloid dendritic cell (pDC and mDC) homing receptors were increased, but the maturation of mDCs, in particular of CD16+ mDCs, was more important than that of pDCs. Monitoring of 15 cytokines showed that those, which are known to be increased early in HIV-1/SIVmac pathogenic infections, such as IL-15, IFN-α, MCP-1 and CXCL10/IP-10, were significantly increased in AGMs as well. In contrast, cytokines generally induced in the later stage of acute pathogenic infection, such as IL-6, IL-18 and TNF-α, were less or not increased, suggesting an early control of IA. We then treated AGMs daily with high doses of IFN-α from day 9 to 24 post-infection. No impact was observed on the activation or maturation profiles of mDCs, pDCs and NK cells. There was also no major difference in T cell activation or interferon-stimulated gene (ISG) expression profiles and no sign of disease progression. Thus, even after administration of high levels of IFN-α during acute infection, AGMs were still able to control IA, showing that IA control is independent of IFN-α levels. This suggests that the sustained ISG expression and IA in HIV/SIVmac infections involves non-IFN-α products.
Author Summary
Chronic inflammation is considered as directly involved in AIDS pathogenesis. The role of IFN-α as a driving force of chronic inflammation is under debate. Natural hosts of SIV, such as African green monkeys (AGMs), avoid chronic inflammation. We show for the first time that NK cells are strongly activated during acute SIVagm infection. This further demonstrates that AGMs mount a strong early innate immune response. Myeloid and plasmacytoid dendritic cells (mDCs and pDCs) homed to lymph nodes; however mDCs showed a stronger maturation profile than pDCs. Monitoring of cytokine profiles in plasma suggests that the control of inflammation in AGMs is starting earlier than previously considered, weeks before the end of the acute infection. We tested whether the capacity to control inflammation depends on the levels of IFN-α produced. When treated with high doses of IFN-α during acute SIVagm infection, AGMs did not show increase of immune activation or signs of disease progression. Our study provides evidence that the control of inflammation in SIVagm infection is not the consequence of weaker IFN-α levels. These data indicate that the sustained interferon-stimulated gene induction and chronic inflammation in HIV/SIVmac infections is driven by factors other than IFN-α.
doi:10.1371/journal.ppat.1004241
PMCID: PMC4081777  PMID: 24991927
18.  Inflammatory mechanisms in the lung 
Inflammation is the body’s response to insults, which include infection, trauma, and hypersensitivity. The inflammatory response is complex and involves a variety of mechanisms to defend against pathogens and repair tissue. In the lung, inflammation is usually caused by pathogens or by exposure to toxins, pollutants, irritants, and allergens. During inflammation, numerous types of inflammatory cells are activated. Each releases cytokines and mediators to modify activities of other inflammatory cells. Orchestration of these cells and molecules leads to progression of inflammation. Clinically, acute inflammation is seen in pneumonia and acute respiratory distress syndrome (ARDS), whereas chronic inflammation is represented by asthma and chronic obstructive pulmonary disease (COPD). Because the lung is a vital organ for gas exchange, excessive inflammation can be life threatening. Because the lung is constantly exposed to harmful pathogens, an immediate and intense defense action (mainly inflammation) is required to eliminate the invaders as early as possible. A delicate balance between inflammation and anti-inflammation is essential for lung homeostasis. A full understanding of the underlying mechanisms is vital in the treatment of patients with lung inflammation. This review focuses on cellular and molecular aspects of lung inflammation during acute and chronic inflammatory states.
PMCID: PMC3218724  PMID: 22096348
inflammation; lung; inflammatory mediators; cytokines
19.  Fetal Inflammatory Response in Women with Proteomic Biomarkers Characteristic of Intra-Amniotic Inflammation and Preterm Birth 
Objective
To determine the relationship between presence of amniotic fluid (AF) biomarkers characteristic of inflammation (defensins 2 and 1, calgranulins C and A) and fetal inflammatory status at birth.
Design
Prospective observational cohort.
Setting
Tertiary referral University hospital
Population
132 consecutive mothers (gestational age, median [interquartile range]: 29.6 [24.1-33.6] weeks), who had a clinically indicated amniocentesis to rule-out infection and their newborns.
Methods
Intra-amniotic inflammation was diagnosed by mass spectrometry SELDI-TOF. The AF proteomic fingerprint [Mass Restricted (MR) score] ranges from 0-4 (none to all biomarkers present). The intensity of intra-amniotic inflammation was graded based on the number of proteomic biomarkers: MR score 0: “no” inflammation; MR score 1-2: “minimal” inflammation; MR score 3-4: “severe” inflammation. At birth, cord blood was obtained for all cases. Severity of histological chorioamnionitis (HCA) and early onset neonatal sepsis (EONS) was based on established histological and hematological criteria. Interleukin-6 (IL-6) levels were measured by sensitive immunoassays. The cord blood-to-AF IL-6 ratio was used as an indicator of the differential inflammatory response in the fetal versus the AF compartment.
Main Outcome Measures
to relate proteomic biomarkers of intra-amniotic infection to cord blood IL-6 and to use the latter as the primary marker of fetal inflammatory response.
Results
Women with intra-amniotic inflammation delivered at an earlier gestational age (ANOVA, P<0.001) and had higher AF IL-6 levels (P<0.001). At birth, neonates of women with “severe” intra-amniotic inflammation had higher cord blood IL-6 levels (P=0.002) and a higher frequency of EONS (P=0.002). EONS was characterized by significantly elevated cord blood IL-6 levels (P<0.001). Out of the 39 neonates delivered by mothers with “minimal” intra-amniotic inflammation, 15 (39%) had umbilical cord blood IL-6 levels above the mean for the group, and 2 neonates had confirmed sepsis. The severity of the neutrophilic infiltrate in the chorionic plate (P<0.001), choriodecidua (P=0.002), umbilical cord (P<0.001), but not amnion (P>0.05) was an independent predictor of the cord blood-to-AF IL-6 ratio. Relationships were maintained following correction for gestational age, birthweight, amniocentesis-to delivery interval, cesarean delivery, status of the membranes, race, MR score, antibiotics and steroid exposure.
Conclusion
We provide evidence that presence in the AF of proteomic biomarkers characteristic of inflammation is associated with an increased inflammatory status of the fetus at birth. Neonates mount an increased inflammatory status and have positive blood cultures even in the context of “minimal” intra-amniotic inflammation.
doi:10.1111/j.1471-0528.2008.01925.x
PMCID: PMC3791329  PMID: 18947340
Proteomics; biomarkers; amniotic fluid; inflammation; defensin; calgranulin; umbilical cord blood; interleukin-6
20.  STUDIES ON THE PATHOGENESIS OF STAPHYLOCOCCAL INFECTION  
Studies have been described in which the effect of early and late or established inflammation, upon staphylococcus infection of rabbit skin has been evaluated. Inflammation was produced in skin by thermal, chemical, bacterial and immunological injury, and it was found that the area of inflammation was more susceptible to staphylococcus infection than was normal skin if the bacteria were injected within 2 to 3 days after the injury. When staphylococci were injected into an area of inflammation of over 3 days' duration, there appeared to be an increase in local resistance to infection. The way in which inflammation was produced seemed to have a little influence upon the effects observed. This influence of non-specific inflammation upon staphylococcus infection was compared with the influence of specific bacterial hypersensitivity, which also is associated with an increase in infectivity of the microorganism in sensitized animals. It was concluded that specific bacterial hypersensitivity probably increases susceptibility to infection with the staphylococcus in the same way as non-specific inflammation. The general significance of non-specific inflammation upon infection is also discussed.
PMCID: PMC2137348  PMID: 13707352
21.  Apoptosis Is Essential for Neutrophil Functional Shutdown and Determines Tissue Damage in Experimental Pneumococcal Meningitis 
PLoS Pathogens  2009;5(5):e1000461.
During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1β and G-CSF as well as reduced levels of anti-inflammatory TGF-β. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils.
Author Summary
Infections are typically accompanied by inflammation and the presence of leucocytes at the infected site. Especially in pyogenic (pus generating) infections, neutrophils are the main leukocytes that arrive and that combat the infection. When the bacteria have been killed, the inflammation also has to be resolved, and this resolution involves the death of the neutrophils through the process of apoptosis. Although we know that neutrophils eventually die through apoptosis, the connection between neutrophil apoptosis and inflammation in bacterial infections has not been clear. In meningitis, bacteria gain access to the space surrounding the brain, multiply and cause inflammation. Despite antibiotic therapy, human pneumococcal meningitis often causes brain damage and death of the patient. We show here that the experimental inhibition of neutrophil apoptosis leads to much more severe clinical disease in a mouse model of bacterial (pneumococcal) meningitis. This was due to a prolonged inflammatory activity of the neutrophils. Experimental induction of neutrophil apoptosis in mice that had been infected and treated with antibiotics (mimicking human meningitis) improved the clinical condition and accelerated recovery. Experimental manipulation of neutrophil apoptosis can therefore be beneficial in acute inflammation.
doi:10.1371/journal.ppat.1000461
PMCID: PMC2682662  PMID: 19478887
22.  Impaired Integrity of DNA after Recovery from Inflammation Causes Persistent Dysfunction of Colonic Smooth Muscle 
Gastroenterology  2011;141(4):1293-1301.e3.
Background & Aims
Patients with inflammatory bowel disease who are in remission and those that developed inflammatory bowel syndrome after enteric infection continue to have symptoms of diarrhea or constipation in the absence of overt inflammation, indicating motility dysfunction. We investigated whether oxidative stress during inflammation impairs integrity of the promoter of Cacna1c, which encodes the pore-forming α1C subunit of Cav1.2b calcium channels.
Methods
We used long-extension PCR (LX-PCR) to evaluate DNA integrity in tissues from distal colons of rats; trinitrobenzene sulfonic acid (TNBS) was used to induce inflammation.
Results
H2O2 increased in the muscularis externa 1 to 7 days after inflammation was induced with TNBS. The oxidative stress significantly impaired DNA integrity in 2 specific segments of the Cacna1c promoter: −506 to −260 and −2,193 to −1,542. The impairment peaked at day 3 and recovered partially by day 7 after induction of inflammation; expression of the products of Cacna1c followed a similar time course. Oxidative stress suppressed the expression of Nrf2, an important regulator of anti-oxidant proteins. Intra-peritoneal administration of sulforaphane significantly reversed the suppression of Nrf2, oxidative damage in the promoter of Cacna1c, and suppression of Cacna1c on day 7 of inflammation. The inflammation subsided completely by 56 days after inflammation was induced; however, impairment of DNA integrity, expression of Nrf2 and Cacna1c, and smooth muscle reactivity to acetylcholine remained suppressed at this timepoint.
Conclusion
Oxidative stress during inflammation impairs the integrity of the promoter of Cacna1c; impairment persists partially after inflammation has subsided. Reduced transcription of Cacna1c contributes to smooth muscle dysfunction in the absence of inflammation.
doi:10.1053/j.gastro.2011.06.074
PMCID: PMC3186840  PMID: 21745450
IBD; IBS; gastrointestinal inflammation; DNA repair; damage; mutation
23.  Junctional Adhesion Molecule-A Regulates Vascular Endothelial Growth Factor Receptor-2 Signaling-Dependent Mouse Corneal Wound Healing 
PLoS ONE  2013;8(5):e63674.
Inflammation and angiogenesis are integral parts of wound healing. However, excessive and persistent wound-induced inflammation and angiogenesis in an avascular tissue such as the cornea may be associated with scarring and visual impairment. Junctional adhesion molecule A (Jam-A) is a tight junction protein that regulates leukocyte transmigration as well as fibroblast growth factor-2 (FGF-2)-induced angiogenesis. However its function in wound-induced inflammation and angiogenesis is still unknown. In this study, we report spontaneous corneal opacity in Jam-A deficient mice associated with inflammation, angiogenesis and the presence of myofibroblasts. Since wounds and/or corneal infections cause corneal opacities, we tested the role of Jam-A in wound-induced inflammation, angiogenesis and scarring by subjecting Jam-A deficient mice to full thickness corneal wounding. Analysis of these wounds demonstrated increased inflammation, angiogenesis, and increased number of myofibroblasts thereby indicating that Jam-A regulates the wound-healing response by controlling wound-induced inflammation, angiogenesis and scarring in the cornea. These effects were not due to inflammation alone since the inflammation-induced wound-healing response in Jam-A deficient mice was similar to wild type mice. In order to determine the molecular mechanism associated with the observed aberrant corneal wound healing in Jam-A deficient mice, we assessed the expression of the components of vascular endothelial growth factor A (VEGF-A)/vascular endothelial growth factor receptor- 2(VEGFR-2) signaling pathway. Interestingly, we observed increased levels of VEGF-A mRNA in Jam-A deficient eyes. We also observed nuclear localization of phosphorylated SMAD3 (pSMAD3) indicative of TGFβ pathway activation in the Jam-A deficient eyes. Furthermore the increased wound-induced corneal inflammation, angiogenesis, and scarring in Jam-A deficient mice was attenuated by treatment with DC101, an anti-vascular endothelial growth factor receptor-2 (VEGFR-2) antibody. Our results suggest that in the absence of Jam-A, the VEGF-A/VEGFR-2 pathway is upregulated, thereby augmenting wound induced corneal inflammation, angiogenesis, and myofibroblast accumulation leading to scarring.
doi:10.1371/journal.pone.0063674
PMCID: PMC3648504  PMID: 23667656
24.  Inflammation Fuels Colicin Ib-Dependent Competition of Salmonella Serovar Typhimurium and E. coli in Enterobacterial Blooms 
PLoS Pathogens  2014;10(1):e1003844.
The host's immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae (“inflammation-inflicted blooms”). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family.
Author Summary
Colicins are bacterial protein toxins which show potent activity against sensitive strains in vitro. Ecological models suggest that colicins play a major role in modulating dynamics of bacterial populations in the gut. However, previous studies could not readily confirm these predictions by respective in vivo experiments. In animal models, colicin-producing strains only show a minor or even absent fitness benefit over sensitive competitors. Here, we propose that the gut environment plays a crucial role in generating conditions for bacterial competition by colicin Ib (ColIb). Gut inflammation favours overgrowth of Enterobacteriaceae (“inflammation-inflicted Enterobacterial blooms”). We show that a pathogenic Salmonella Typhimurium (S. Tm) strain benefits from ColIb production in competition against commensal E. coli upon growth in inflammation-inflicted blooms. In the absence of gut inflammation, ColIb production did not confer a competitive advantage to S. Tm. In the inflamed gut, the genes for ColIb production in S. Tm and its corresponding ColIb-surface receptor CirA in E. coli were markedly induced, as compared to the non-inflamed gut. Therefore, environmental conditions in inflammation-inflicted blooms favour colicin-dependent competition of Enterobacteriaceae by triggering ColIb production and susceptibility at the same time. Our findings reveal a role of colicins as important bacterial fitness factors in inflammation-induced blooms.
doi:10.1371/journal.ppat.1003844
PMCID: PMC3879352  PMID: 24391500
25.  Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis 
PLoS Pathogens  2010;6(4):e1000841.
Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.
Author Summary
Adenoviruses are nonenveloped DNA viruses that infect mucosal tissues, causing a wide array of diseases. Adenovirus infection of the cornea induces inflammation in the form of multifocal leukocytic infiltrates. Although studied extensively in tissue culture models, how adenoviruses induce inflammation in the living host is not well characterized in the cornea or elsewhere. Using a unique mouse model, we studied the role of viral components in the cornea, to determine which viral part(s) induce an innate immune response. We found that neither viral DNA or viral gene expression was necessary for the development of inflammation. In contrast, viral capsid, the protein coat of the virus, induced inflammation similar to intact virus. Mice lacking the toll-like receptor 9 (Tlr9) molecule, which acts as a pathogen DNA-sensing molecule within the cell, developed clinical inflammation upon adenovirus infection similar to wild type mice. Virus associated inflammation in the mouse cornea could be blocked by treatment with a peptide containing components of the adenoviral capsid. Adenovirus infection of the cornea induces inflammation principally through contact between the viral capsid and the host cell. Our study provides new insights into how the innate immune system in the eye responds to a clinically important viral pathogen.
doi:10.1371/journal.ppat.1000841
PMCID: PMC2855317  PMID: 20419141

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