Tissue Bacterial Load Research Articles

The protection effect of LEAP-2 on the mudskipper (Boleophthalmus pectinirostris) against Edwardsiella tarda infection is associated with its immunomodulatory activity on monocytes/macrophages

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic peptide that plays an important role in the host's innate immune system. However, the mechanism by which LEAP-2 modulates/regulates the host defense against pathogens remains largely unknown. In this study, we identified a cDNA sequence encoding LEAP-2 homolog (BpLEAP-2) in the mudskipper, Boleophthalmus pectinirostris. Sequence analysis revealed that BpLEAP-2 belonged to the fish LEAP-2A cluster and that it was closely related to ayu LEAP-2. BpLEAP-2 mRNA was detected in a wide range of tissues, with the highest level of transcripts found in the liver. Upon infection with Edwardsiella tarda, BpLEAP-2 mRNA expression was significantly increased in the liver, kidney, spleen, and gill, but decreased in the intestine. Chemically synthesized BpLEAP-2 mature peptide did not exhibit antibacterial activity against E. tarda in vitro. Intraperitoneal injection of BpLEAP-2 (1.0 or 10.0 μg/g) resulted in significantly improved survival rate and reduced tissue bacterial load in E. tarda-infected mudskippers. In E. tarda-infected fish, BpLEAP-2 (0.1, 1.0, or 10.0 μg/g) eliminated E. tarda-induced tissue mRNA expression of BpTNF-α and BpIL-1β. In monocytes/macrophages (MO/MФ), BpLEAP-2 (1.0 or 10.0 μg/ml) induced chemotaxis, enhanced respiratory burst, and inhibited E. tarda-induced mRNA expression of BpTNF-α and BpIL-1β. At a concentration of 10.0 μg/ml, BpLEAP-2 also significantly enhanced the bacterial killing efficiency of MO/MФ. No significant effect was seen in the phagocytic activity of MO/MФ upon treatment with BpLEAP-2. Our study provides evidence, for the first time, that LEAP-2 exhibited immunomodulatory effects on immune cells, and protected the host from pathogenic infections independent of direct bacterial killing function.

Molecular characterization of an interleukin-4/13B homolog in grass carp (Ctenopharyngodon idella) and its role in fish against Aeromonas hydrophila infection

Mammalian interleukin 4 (IL-4) and interleukin 13 (IL-13) molecules are anti-inflammatory cytokines mediating the alternative activation of macrophages. However, the role of fish IL-4/13 homologs in monocytes/macrophages (MO/MФ) polarization remains unclear. In this study, we have functionally identified an IL-4/13B homolog in grass carp (Ctenopharyngodon idella), which is termed as CiIL-4/13B. Multiple alignment showed that CiIL-4/13B shared the typical characteristics and structure with other known fish IL-4/13. Phylogenetic analysis showed that CiIL-4/13B is evolutionarily closely related to zebrafish (Danio rerio) and common carp (Cyprinus carpio) IL-4/13B. CiIL-4/13B mRNA was constitutively expressed in tissues and peripheral blood lymphocytes (PBLs) examined, with its highest expression seen in PBLs. Following Aeromonas hydrophila infection, CiIL-4/13B mRNA expression was upregulated. Recombinant CiIL-4/13B (rCiIL-4/13B) was overexpressed in Escherichia coli and purified for a functional study. Using prepared anti-rCiIL-4/13B antiserum, Western blot analysis showed that native CiIL-4/13B in grass carp plasma is N-glycosylated. Intraperitoneal injection of bioactive rCiIL-4/13B significantly increased the survival rate of grass carp against A. hydrophila, and decreased the tissue bacterial load, with a higher dose having better effects. Bioactive rCiIL-4/13B treatment decreased nitrite production and mRNA expression of proinflammatory cytokines (IL-1β and TNF-α), while it increased arginase activity and mRNA expression of anti-inflammatory cytokines (TGF-β and IL-10). The phagocytosis by grass carp MO/MФ had no significant changes by the 8 h treatment of bioactive rCiIL-4/13B compared to that of the negative control, while it was significantly inhibited by the 24 h treatment of bioactive rCiIL-4/13B. The inhibitory effect of rCiIL-4/13B on MO/MФ phagocytosis may be a consequence of MO/MФ proliferation. In summary, our results suggest that CiIL-4/13B plays a protective effect in grass carp against A. hydrophila by inducing alternatively activated MO/MФ.

Type I interferon promotes alveolar epithelial type II cell survival during pulmonary Streptococcus pneumoniae infection and sterile lung injury in mice.

Protecting the integrity of the lung epithelial barrier is essential to ensure respiration and proper oxygenation in patients suffering from various types of lung inflammation. Type I interferon (IFN-I) has been associated with pulmonary epithelial barrier function, however, the mechanisms and involved cell types remain unknown. We aimed to investigate the importance of IFN-I with respect to its epithelial barrier strengthening function to better understand immune-modulating effects in the lung with potential medical implications. Using a mouse model of pneumococcal pneumonia, we revealed that IFN-I selectively protects alveolar epithelial type II cells (AECII) from inflammation-induced cell death. Mechanistically, signaling via the IFN-I receptor on AECII is sufficient to promote AECII survival. The net effects of IFN-I are barrier protection, together with diminished tissue damage, inflammation, and bacterial loads. Importantly, we found that the protective role of IFN-I can also apply to sterile acute lung injury, in which loss of IFN-I signaling leads to a significant reduction in barrier function caused by AECII cell death. Our data suggest that IFN-I is an important mediator in lung inflammation that plays a protective role by antagonizing inflammation-associated cell obstruction, thereby strengthening the integrity of the epithelial barrier.

Antibacterial efficacy and pharmacokinetic evaluation of sanguinarine in common carp (Cyprinus carpio) following a single intraperitoneal administration

Sanguinarine (SA), with antimicrobial and antiparasitic activities against fish pathogens, exhibits great potential commercial use in aquaculture. However, little information on pharmacokinetics of SA restricts further application in aquaculture. In this study, pharmacokinetics of SA in common carp (Cyprinus carpio) following a single intraperitoneal administration [10mgkg(-1) BW (body weight)] was evaluated by high-performance liquid chromatography (HPLC). The peak concentration (Cmax ) of SA in kidney was 11.8μgg(-1) , which was higher than in other tissues and plasma. The terminal half-life in fish tissue and plasma was as follows: 42.3h (kidney)>37.2h (liver)>20.1h (gill)>18.8h (muscle)>10.9h (spleen)>10.0h (plasma). Additionally, we determined the bacterial loads in tissues of common carp infected with Aeromonas hydrophila after i.p. administration of SA at 0, 5, 10 and 20mgkg(-1) BW. The results showed that i.p. administration of SA at 10mgkg(-1) BW significantly enhanced antibacterial efficacy against A.hydrophila, where the antibacterial ratio in the gill, kidney, spleen and liver on day 5 was 95.13%, 93.33%, 90.09% and92.82%, respectively. Overall, these results suggested the potential of SA to treat A.hydrophila infection in common carp farming industry.

Development of an Orientia tsutsugamushi Lc-1 Murine Intraperitoneal Challenge Model for Scrub Typhus: Determination of Murine Lethal Dose (MuLD50), Tissue Bacterial Loads, and Clinical Outcomes.

Currently, no vaccine has been developed to protect humans from naturally acquired heterologous Orientia tsutsugamushi infections. To enhance the validity of vaccine candidates, we are developing a murine chigger challenge model with the O. tsutsugamushi Lc-1-infected Leptotrombidium chiangraiensis Line-1. To this end, an intraperitoneal (i.p.) murine challenge model using an O. tsutsugamushi Lc-1 isolate was developed for eventual validation of the chigger challenge model. We have determined that the murine lethal dose that kills 50% of the challenged mice (MuLD50) of a liver/spleen homogenate developed from O. tsutsugamushi Lc-1-infected ICR Swiss mice to be 10(-6.9). Employing different inoculum doses of this homogenate, the bacterial load using quantitative real-time PCR (qPCR) was determined to range from 60 to 1.6 × 10(5) genome equivalent copies (GEC)/μL of liver and 33.4 to 2.2 × 10(5) GEC/μL of spleen tissue. The clinical outcomes relative to homogenate dose levels followed a dose-dependent pattern. The successful development and characterization of the O. tsutsugamushi Lc-1 i.p. challenge model will assist in the development and validation of a mouse chigger challenge scrub typhus model.

Increased ferroportin-1 expression and rapid splenic iron loss occur with anemia caused by Salmonella enterica Serovar Typhimurium infection in mice.

The Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium causes persistent systemic inflammatory disease in immunocompetent mice. Following oral inoculation with S. Typhimurium, mice develop a hematopathological syndrome akin to typhoid fever with splenomegaly, microcytic anemia, extramedullary erythropoiesis, and increased hemophagocytic macrophages in the bone marrow, liver, and spleen. Additionally, there is marked loss of iron from the spleen, an unanticipated result, given the iron sequestration reported in anemia of inflammatory disease. To establish why tissue iron does not accumulate, we evaluated multiple measures of pathology for 4 weeks following oral infection in mice. We demonstrate a quantitative decrease in splenic iron following infection despite increased numbers of splenic phagocytes. Infected mice have increased duodenal expression of the iron exporter ferroportin-1, consistent with increased uptake of dietary iron. Liver and splenic macrophages also express high levels of ferroportin-1. These observations indicate that splenic and hepatic macrophages export iron during S. Typhimurium infection, in contrast to macrophage iron sequestration observed in anemia of inflammatory disease. Tissue macrophage export of iron occurs concurrent with high serum concentrations of interferon gamma (IFN-γ) and interleukin 12 (IL-12). In individual mice, high concentrations of both proinflammatory (tumor necrosis factor alpha [TNF-α]) and anti-inflammatory (IL-10) cytokines in serum correlate with increased tissue bacterial loads throughout 4 weeks of infection. These in vivo observations are consistent with previous cell culture studies and suggest that the relocation of iron from tissue macrophages during infection may contribute to anemia and also to host survival of acute S. Typhimurium infection.

Improved oral therapeutic potential of nanoencapsulated cryptdin formulation against Salmonella infection

An encapsulated system for cryptdin-2 (a Paneth cell antimicrobial peptide) was developed, with a view to help it sustain adverse gut conditions and to ensure its bioavailability on oral administration. The formulation was characterized on the basis of particle size, zeta potential and polydispersity index. Cryptdin-2 loaded nanoparticles of size 105±7nm, formulated by ionotropic gelation method using chitosan: tripolyphosphate (5:2), revealed 60% drug entrapment efficiency with 65% in vitro release in 4.5h. Developed system was evaluated for its therapeutic application against Salmonella Typhimurium infection in mice, on the basis of survivability of animals, bacterial load in tissues, histo-architecture and oxidative damage markers. Infected mice when treated with the encapsulated peptide showed 83% survivability and approximately 2 log unit reductions in the bacterial load in the tissues versus 100% mortality observed with the free peptide. The encapsulated cryptdin-2 also achieved a decrease in the level of oxidants, particularly nitrite by 3.25 folds and increased the level of antioxidant catalase by 2 folds when compared to the levels exhibited by the free peptide. The bacteriological and biochemical alterations illustrated by encapsulated peptide co-related well with the histo-architectural studies. The study is a first pre-clinical report on the oral effectiveness of cryptdin-2 by its suitable encapsulation and has potential for future clinical applications.

The relationships between Brucella melitensis predilection sites, bacterial loads in vivo, and the agglutinating antibody response in experimentally infected sheep

F?or exploring Brucella melitensis survival in in vivo predilection sites and the relationship between bacterial loads and the detection of antibody titers in experimentally subcutaneously infected sheep with B.1397645907melitensis 16M, ten rams and ten ewes were used. One ram and one ewe were euthanized at 7, 15, 30, 60, 90, 120, and 180 days post inoculation (dpi). Bacteriological results showed that tissue isolation rates and bacterial loads peaked from 7 dpi to 30 dpi and then cleared until 120 dpi. In in situ hybridization trials the strongest signals for B.1397645907melitensis were detected at 7 dpi and 15 dpi, and then they gradually cleared. Monitoring results of agglutinating antibodies showed that anti-Brucella antibodies began to be produced at 7 dpi, peaked at 15 dpi, and gradually declined until lower detection levels than the threshold were being detected at 180 dpi. Thus, we found that the more Brucella bacterial loads there were in vivo, the higher the antibody titers were in sera, which suggested that detection of the agglutinating antibody titers might be used as an indicator of a Brucella carrier in infected animals.

Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection.

The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010), one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (<1.0 endotoxin unit/106 cells) compared to wild type cells. Immunization with formalin inactivated IB010 produced a robust antibody response consisting of both IgG1 and IgG2c subtypes. Mice immunized with IB010 had significantly lower post-infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.

Nonclassical MHC Ib-restricted CD8+ T cells recognize Mycobacterium tuberculosis-derived protein antigens and contribute to protection against infection (MPF6P.744)

Abstract MHC Ib-restricted CD8+ T cells have been implicated to contribute to host defense during Mycobacteria tuberculosis (Mtb) infection, but the relative contribution of various MHC Ib-restricted responses has yet to be defined. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-, DKO), MHC Ia/M3 (Kb-/-Db-/-M3-/-, TKO), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb infection. Unlike their role in Listeria infection, we found that M3-restricted CD8+ T cells only represent a small proportion of the CD8+ T cells responding to Mtb infection. This finding highlights the differential role of various MHC Ib-restricted responses in immunity against distinct microbial pathogens. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of TKO mice and expressed higher level of KLRG1 compared to MHC Ia-restricted CD8+ T cells. These CD8+ T cells produced IFNγ, TNFα and IL-2 upon stimulation with Mtb-derived protein antigens in a β2m-dependent but TAP-independent manner. TKO mice had similar bacterial loads in tissues as DKO and wildtype mice, but lower bacterial loads than β2m-/- mice. Our findings indicate non-M3, MHC Ib-restricted CD8+ T cells can specifically recognize Mtb protein antigens and contribute to host immune response to Mtb. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better vaccines against Mtb, due to the limited polymorphism of MHC class Ib molecules.

Neutrophils in granulomas from Mycobacterium tuberculosis-infected macaques express granzyme B in response to mycobacterial products and pro-inflammatory signals (INC7P.406)

Abstract Inflammation is a critical component of tuberculosis (TB), and may contribute to a poor prognosis. The factors responsible for this inflammation are poorly studied. In addition, the role of neutrophils in TB, and whether neutrophils express granzyme B (grzB), a cytotoxic pro-apoptosis enzyme, is controversial. We examined neutrophils in peripheral blood (PB) and granulomas to determine if mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. PB neutrophils did not express grzB. In contrast, granuloma neutrophils expressed grzB and these cells contained more grzB than T cells. Elevated neutrophil, but not T cell, grzB expression correlated with higher tissue bacterial load. Perforin is required for T cell-mediated cytotoxicity but was not expressed by neutrophils. PB neutrophils upregulated grzB expression when infected with M. tuberculosis or stimulated with M. tuberculosis culture filtrate protein or LPS from E. coli, but not peptide antigens associated with T cell stimulation. Moreover, stimulated PB neutrophils secreted grzB in a perforin-independent process. GrzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration or regulating activation in other cell types. These data indicate M. tuberculosis-specific factors in granulomas upregulate neutrophil grzB expression and suggest a previously unappreciated aspect of neutrophil biology in TB.

Immunomodulatory Intervention with Interferon-γ in Escherichia coli Pyelonephritis

We investigated the efficacy of recombinant human interferon-γ in experimental pyelonephritis due to Escherichia coli. Pyelonephritis was induced by intrapelvic inoculation ofbacteria after ureteral ligation in 38 rabbits assigned to 1 of 3 groups, including group 1-16 controls, group 2-14 rabbits treated with intravenous recombinant human interferon-γ and group 3-8 rabbits treated with intravenous recombinant human interferon-γ plus amikacin. Bacterial counts, cytokines and malondialdehyde were measured in blood. Peripheral blood mononuclear cells were isolated to measure TNFα transcripts, cytokine stimulation and apoptosis. Survival was recorded, and the tissue bacterial load and myeloperoxidase activity were measured after sacrifice. The mortality rate in groups 1, 2 and 3 was 66.7%, 25% and 12.5%, respectively. The circulating bacterial count and tissue bacterial load were less in group 2 than in group 1. Circulating malondialdehyde negatively correlated with the bacterial load of the spleen. Although the number of TNFα transcripts in circulating peripheral blood mononuclear cells did not differ, peripheral blood mononuclear cells isolated from group 2 at 48 hours produced much greater concentrations of tumor necrosis factor-α after stimulation with Pam3Cys. In parallel, the apoptosis rate of circulating monocytes was increased in group 2 at48 hours. Lung myeloperoxidase activity at 24 hours, serving as indirect evidence of neutrophil infiltration, was decreased in group 2. Recombinant human interferon-γ administration prolonged survival in rabbits with experimental E. coli urosepsis. Its action was probably related to increased bacterial phagocytosis after modulation of oxidant status and reversal of monocyte immunoparalysis.

Asymptomatic Cattle Naturally Infected with Mycobacterium bovis Present Exacerbated Tissue Pathology and Bacterial Dissemination

Rational discovery of novel immunodiagnostic and vaccine candidate antigens to control bovine tuberculosis (bTB) requires knowledge of disease immunopathogenesis. However, there remains a paucity of information on the Mycobacterium bovis-host immune interactions during the natural infection. Analysis of 247 naturally PPD+ M. bovis-infected cattle revealed that 92% (n = 228) of these animals were found to display no clinical signs, but presented severe as well as disseminated bTB-lesions at post-mortem examination. Moreover, dissemination of bTB-lesions positively correlated with both pathology severity score (Spearman r = 0.48; p<0.0001) and viable tissue bacterial loads (Spearman r = 0.58; p = 0.0001). Additionally, granuloma encapsulation negatively correlated with M. bovis growth as well as pathology severity, suggesting that encapsulation is an effective mechanism to control bacterial proliferation during natural infection. Moreover, multinucleated giant cell numbers were found to negatively correlate with bacterial counts (Spearman r = 0.25; p = 0.03) in lung granulomas. In contrast, neutrophil numbers in the granuloma were associated with increased M. bovis proliferation (Spearman r = 0.27; p = 0.021). Together, our findings suggest that encapsulation and multinucleated giant cells control M. bovis viability, whereas neutrophils may serve as a cellular biomarker of bacterial proliferation during natural infection. These data integrate host granuloma responses with mycobacterial dissemination and could provide useful immunopathological-based biomarkers of disease severity in natural infection with M. bovis, an important cattle pathogen.

Effects of paratuberculosis on Friesian cattle carcass weight and age at culling

Bovine paratuberculosis (PTB) causes major economic losses to dairy farmers because of decreased milk production, poor body condition, weight loss and early culling. The aim of this study was to evaluate the effect of Mycobacterium avium subspecies paratuberculosis (MAP) infection on carcass weight and age at slaughter in Friesian cattle. A total of 1,014 adult cows slaughtered at two local abattoirs in the Basque Country were included in this study. MAP infection was determined by different methods: indirect ELISA on blood samples, detection of MAP in tissues by culture and real time PCR (rtPCR), and histopathological examination. Serial and parallel interpretations of these methods were also considered for setting the upper and lower infection rates. MAP infection was confirmed by at least one test in 58.9% of animals. Most infected cows were detected by histopathology (46.9%) and rtPCR (29.6%). Overall estimates of carcass mean weight losses ranged from 3.7% for cases identified by the presence of microscopic specific PTB inflammatory lesions to 12.4% for cases with positive results in the paratuberculosis antibody ELISA test. Isolation of high bacterial loads in tissues and occurrence of diffuse granulomatous enteritis were associated with the highest weight loses, 22.2% and 26.0% respectively. The life expectancy of seropositive cows and those showing diffuse lesions was reduced by nearly one year compared to that of non-infected ones. Our results provide consistent evidences of PTB effect on the reduction of slaughter weight and lifespan of dairy cows, which could be considered as surrogates of clinical disease.

The Role of B Cells in the Immune Response to Mycobacterium tuberculosis. (43.3)

Abstract The role of humoral immunity in the immune response to Mycobacterium tuberculosis (Mtb) is poorly defined. B cells form aggregates with features of germinal centers and are prominent components of the lung granuloma in Mtb-infected humans, mice and macaques. Using muMT B cell-deficient mice, we showed that B cells were required for optimal granulomatous response and protection upon aerosol infection. muMT mice were more susceptible to Mtb with increased mortality and elevated lung bacterial burden. B cell-deficiency also resulted in exacerbated immunopathology with increased neutrophil influx and IL-10 production independent of tissue bacterial load. Transfer of B cells reversed the phenotypes of the muMT mice. This occurred in the presence of immunoglobulins (Igs) in the serum but without the presence of lung B cells. We showed that engagement of distinct Fcγ receptors (FcγR) divergently affected susceptibility to Mtb. Mice lacking activating FcγR showed increased mortality and bacterial load with enhanced lung neutrophil influx and IL-10 expression compared to wildtype, whereas mice lacking inhibitory FcγRIIB exhibited an augmented lung Th1 response. Thus, B cells via their ability to produce Igs may regulate T cell responses by immune complex engagement of FcγR on antigen presenting cells. The mechanisms by which B cells modulate the immune response to Mtb are being characterized by testing the immune response of transgenic mice deficient in B cells or Igs to infection.

A Novel Tuberculosis DNA Vaccine in an HIV-1 p24 Protein Backbone Confers Protection against Mycobacterium tuberculosis and Simultaneously Elicits Robust Humoral and Cellular Responses to HIV-1

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major infectious disease worldwide. Moreover, latent M. tuberculosis infection is more likely to progress to active TB and eventually leads to death when HIV infection is involved. Thus, it is urgent to develop a novel TB vaccine with immunogenicity to both M. tuberculosis and HIV. In this study, four uncharacterized T cell epitopes from MPT64, Ag85A, Ag85B, and TB10.4 antigens of M. tuberculosis were predicted, and HIV-1-derived p24, an immunodominant protein that can induce protective responses to HIV-1, was used as an immunogenic backbone. M. tuberculosis epitopes were incorporated separately into the gene backbone of p24, forming a pP24-Mtb DNA vaccine. We demonstrated that pP24-Mtb immunization induced a strong M. tuberculosis-specific cellular response as evidenced by T cell proliferation, cytotoxicity, and elevated frequency of gamma interferon (IFN-γ)-secreting T cells. Interestingly, a p24-specific cellular response and high levels of p24-specific IgG were also induced by pP24-Mtb immunization. When the protective effect was assessed after mycobacterial challenge, pP24-Mtb vaccination significantly reduced tissue bacterial loads and profoundly attenuated the mycobacterial infection-related lung inflammation and injury. Our findings demonstrated that the pP24-Mtb tuberculosis vaccine confers effective protection against mycobacterial challenge with simultaneously elicited robust immune responses to HIV-1, which may provide clues for developing novel vaccines to prevent dual infections.

Systemic Treatment with CpG-B after Sublethal Rickettsial Infection Induces Mouse Death through Indoleamine 2,3-Dioxygenase (IDO)

Due to its strong immune stimulatory effects through TLR9, CpG-containing oligodeoxynucleotides (CpG ODN) have been tested in multiple clinical trials as vaccine adjuvant for infectious diseases and cancer. However, immune suppression induced by systemic administration of CpGs has been reported recently. In this study, we evaluated the impact of CpGs in an acute rickettsiosis model. We found that systemic treatment with type B CpG (CpG-B), but not type A CpG (CpG-A), at 2 days after sublethal R. australis infection induced mouse death. Although wild-type (WT) B6 and IDO−/− mice showed similar survival rates with three different doses of R. australis infection, treatment with CpG-B after sublethal infection consistently induced higher mortality with greater tissue bacterial loads in WT but not IDO−/− mice. Also, CpG-B treatment promoted the development of higher serum concentrations of proinflammatory cytokines/chemokines through IDO. Furthermore, while T cell-mediated immune responses enhanced by CpG-B were independent of IDO, treatment with CpG-B promoted T cell activation, PD-1 expression and cell apoptosis partially through IDO. A depletion study using anti-mPDCA-1 mAb indicated that plasmacytoid dendritic cells (pDC) were not required for CpG-B-induced death of R. australis-infected mice. Additionally, the results in iNOS−/− mice suggested that nitric oxide (NO) was partially involved in CpG-B-induced death of R. australis-infected mice. Surprisingly, pre-treatment with CpG-B before administration of a lethal dose of R. australis provided effective immunity in WT, IDO−/− and iNOS−/− mice. Taken together, our study provides evidence that CpGs exert complex immunological effects by both IDO-dependent and -independent mechanisms, and that systemic treatment with CpGs before or after infection has a significant and distinct impact on disease outcomes.

Reduced Peroxisome Proliferator-Activated Receptor α Expression Is Associated With Decreased Survival and Increased Tissue Bacterial Load in Sepsis

The peroxisome proliferator-activated receptor α (PPAR-α) is a member of the nuclear receptor family with many important physiologic roles related to metabolism and inflammation. Previous research in pediatric patients with septic shock revealed that genes corresponding to the PPAR-α signaling pathway are significantly downregulated in a subgroup of children with more severe disease. In this study, PPAR-α expression analysis using whole-blood derived RNA revealed that PPAR-α expression was decreased in patients with septic shock and that the magnitude of that decrement correlated with the severity of disease. In a mouse model of sepsis, induced by cecal ligation and puncture, knockout mice lacking PPAR-α had decreased survival compared with wild-type animals. Plasma cytokine analysis demonstrated decreased levels of interleukin 1β (IL-1β), IL-6, IL-17, keratinocyte-derived cytokine, macrophage chemoattractant protein 1, macrophage inflammatory protein 2, and tumor necrosis factor α at 24 h in PPAR-α knockout animals. Cell surface markers of activation on splenic dendritic cells, macrophages, and CD8 T cells were reduced in PPAR-α null animals, and the bacterial load in lung and splenic tissues was increased. These data indicate that reduced or absent PPAR-α expression confers a survival disadvantage in sepsis and that PPAR-α plays a role in maintaining appropriate immune functions during the sepsis response.

Outer membrane vesicles as an acellular vaccine against Acinetobacter baumannii

Acinetobacter baumannii produces different types of infections including pneumonia, meningitis, and bloodstream infections. The optimal treatment of these infections has been complicated by the global emergence of multidrug resistant strains, requiring the development of novel approaches for treatment and prevention. Outer membrane vesicles are outpouchings of the bacterial outer membrane that are secreted from numerous pathogenic Gram-negative bacteria. In the present study, we describe the isolation of outer membrane vesicles from A. baumannii and their use as a vaccine in a mouse model of disseminated sepsis. Immunization produced a robust antibody response against multiple bacterial antigens which consisted of antigen-specific IgG and IgM. In addition, both IgG1 and IgG2c subtypes were produced by immunization. Immunized mice had lower tissue bacterial loads and lower serum levels of the pro-inflammatory cytokines IL-6 and IL-1β post-infection compared to control mice. Importantly, vaccination protected mice from challenge with the ATCC 19606 strain and provided protection against two clinical isolates, including a pan-resistant strain. These results indicate that vaccination with outer membrane vesicles may be a viable strategy for preventing A. baumannii infection.

Pandrug-resistant isolate of Klebsiella pneumoniae causes less damage than drug-susceptible isolates in a rabbit model

Bacterial infections induce a series of inflammatory responses and lead to longer hospital stays and increased mortality. In clinical work, we often find that infections caused by drug-susceptible isolates have a worse outcome than those caused by pandrug-resistant isolates. To goal of this study was to assess the impact of drug resistant in a rabbit model of Klebsiella pneumoniae infection. This study used a rabbit model of experimental bacteremia, challenged by susceptible (A), multidrug-resistant (B) and pandrug-resistant (C) isolates of Klebsiella pneumoniae. Mimimal inhibitory concentrations (MIC), leukocyte, TNFα, IL-17, and HMGB-1 levels and survival times were measured. Mean survival times after challenge by isolates A, B and C were 10.5 ± 3.63, 12.7 ± 2.31 and 13.9 ± 0.32 days, respectively. Leukocytes levels after challenge with isolate C were lower compared with those after challenge with isolate A (p = 0.002). Blood counts of the offending pathogens and concentrations of TNFα, IL-17, and HMGB-1 were higher in the group challenged by isolate A in comparison with isolate B or C. Tissue bacterial load after animal death was significantly higher in rabbits of group A in comparison with isolates B and C. Bacteremia induced by pandrug-resistant isolates is accompanied by less damage compared with bacteremia by drug-susceptible isolates. Rabbits infected with a pandrug-resistant isolate of K. pneumoniae survived longer and had a lower inflammatory response than did animals infected with drug-susceptible isolates.