Progressive Control of Streptococcus agalactiae-Induced Innate Inflammatory Response Is Associated with Time Course Expression of MicroRNA-223 by Neutrophils.

Group B streptococcus (GBS) is a human-pathogenic bacterium inducing a strong inflammatory response that may be detrimental for host tissues if not finely regulated. The inflammatory response can be modulated by different molecular mechanisms, among which growing evidence points toward the crucial role of microRNAs (miRNAs). Regarding innate inflammatory response, studies have reported that miR-223 is essential for the control of granulocyte proliferation and activation. Moreover, a number of investigations on miRNA expression profiling performed in various inflammatory settings have revealed that miR-223 is among the top differentially expressed miRNAs. Yet the dynamic pattern of expression of miR-223 in vivo with respect to the evolution of the inflammatory process, especially in microbial infection, remains elusive. In this study, we analyzed the kinetic expression of miR-223 in an inflammatory model of GBS-induced murine pneumonia and looked for correlates with inflammatory markers, including innate cell infiltrates. We found that miR-223 expression is rapidly induced at very early time points (3 to 6 h postinfection [p.i.]) mainly by lung-infiltrating neutrophils. Interestingly, the level of miR-223 accumulating in the lungs remains higher at later stages of infection (24 h and 48 h p.i.), and this correlates with reduced expression of primary inflammatory cytokines and chemokines and with a shift in infiltrating monocyte and macrophage subtypes toward a regulatory phenotype. Transient inhibition of miR-223 by an antagomir resulted in significant increase of CXCL2 expression and partial enhancement of infiltrating neutrophils in GBS-infected lung tissues. This suggests the potential contribution of miR-223 to the resolution phase of GBS-induced acute inflammation.

Single-cell analysis of innate cytokine responses to pattern recognition receptor stimulation in children across four continents.

Innate immunity instructs adaptive immunity, and suppression of innate immunity is associated with an increased risk for infection. We showed previously that whole-blood cellular components from a cohort of South African children secreted significantly lower levels of most cytokines following stimulation of pattern recognition receptors compared with whole blood from cohorts of Ecuadorian, Belgian, or Canadian children. To begin dissecting the responsible molecular mechanisms, we set out to identify the relevant cellular source of these differences. Across the four cohorts represented in our study, we identified significant variation in the cellular composition of whole blood; however, a significant reduction in the intracellular cytokine production on the single-cell level was only detected in South African children's monocytes, conventional dendritic cells, and plasmacytoid dendritic cells. We also uncovered a marked reduction in polyfunctionality for each of these cellular compartments in South African children compared with children from the other continents. Together, our data identify differences in cell composition, as well as profoundly lower functional responses of innate cells, in our cohort of South African children. A possible link between altered innate immunity and increased risk for infection or lower response to vaccines in South African infants needs to be explored.

Sex-biased expression of selected chromosome x-linked microRNAs with potent regulatory effect on the inflammatory response in children with cystic fibrosis: A preliminary pilot investigation.

Previous studies have reported sex disparity in cystic fibrosis (CF) disease, with females experiencing more pulmonary exacerbations and frequent microbial infections resulting in shorter survival expectancy. This concerns both pubertal and prepubertal females, which is in support to the prominent role of gene dosage rather than the hormonal status. The underlying mechanisms are still poorly understood. The X chromosome codes for a large number of micro-RNAs (miRNAs) that play a crucial role in the post-transcriptional regulation of several genes involved in various biological processes, including inflammation. However, their level of expression in CF males and females has not been sufficiently explored. In this study, we compared in male and female CF patients the expression of selected X-linked miRNAs involved in inflammatory processes. Cytokine and chemokine profiles were also evaluated at both protein and transcript levels and cross-analyzed with the miRNA expression levels. We observed increased expression of miR-223-3p, miR-106a-5p, miR-221-3p and miR-502-5p in CF patients compared to healthy controls. Interestingly, the overexpression of miR-221-3p was found to be significantly higher in CF girls than in CF boys and this correlates positively with IL-1ß. Moreover, we found a trend toward lower expression in CF girls than in CF boys of suppressor of cytokine signaling 1 (SOCS1) and the ubiquitin-editing enzyme PDLIM2, two mRNA targets of miR-221-3p that are known to inhibit the NF-κB pathway. Collectively, this clinical study highlights a sex-bias in X-linked miR-221-3p expression in blood cells and its potential contribution to sustaining a higher inflammatory response in CF girls.

Sex difference in innate inflammatory response and macrophage polarization in Streptococcus agalactiae-induced pneumonia and potential role of microRNA-223-3p.

While number of studies have shown that biological sex is a risk factor in the incidence and severity of infection-induced inflammatory diseases, the underlying mechanisms are still poorly understood. In this study, we compared the innate inflammatory response in male and female mice with group B streptococcal (GBS)-induced pneumoniae. Although male and female mice displayed similar bacterial burdens, males exhibited more innate inflammatory cytokines and chemokines and a higher proportion of infiltrating monocytes/macrophages. The analysis of the distribution of macrophage subtypes M1 (pro-inflammatory) versus M2 (anti-inflammatory) yielded a higher M1/M2 ratio in infected males compared with females. Given the importance of the chromosome X-linked microRNA-223-3p (miR-223-3p) in modulating the inflammatory process and macrophage polarization, we investigated its potential contribution in sex bias of GBS-induced innate inflammatory response. Knock-down of miR-223-3p with specific antagomiR resulted in increased inflammatory response and higher M1/M2 ratio following GBS infection. Notably, compared to male mice, we detected higher amount of miR-223-3p in macrophages from females that correlated negatively with M1 phenotype. These results suggest that differential expression of miR-233-3p may impact macrophage polarization, thereby contributing to fine-tune sex differences in inflammatory response.

Immunomodulation using genetically engineered bacteria for type III-mediated delivery of heterologous antigens and cytokines: potential application in vaccine and therapeutical developments.

The rational design of new approaches aiming to improve antigen or drug delivery remains a major challenge in numerous vaccine and therapeutical strategies. Studies on genetically engineered bacteria have led to significant progress in the delivery of heterologous proteins. Yet, the efficiency of such recombinant bacteria relies particularly on their ability to deliver sufficient amount of the drug to intended cell tissues. Gram-negative bacteria like Salmonella, Shigella, Yersinia and Pseudomonas have evolved powerful secretion machinery called type III secretion system (T3SS) that is used to optimize their virulence effect by delivering bacterial effectors to the membrane or into the host cell cytoplasm. This review highlights recent studies unraveling whether T3SS of attenuated bacteria could be an efficient route for delivery of vaccine antigens or bioactive proteins such as cytokines for immunomodulation that could find a broad range of application purposes. These current experimental investigations can be considered proofs of concept that need to be translated readily to effective vaccines and therapeutics in humans.

The Acid-Base Balance and Gender in Inflammation: A Mini-Review.

In humans, acid-base balance is crucial to cell homeostasis. Acidosis is observed in numerous inflammatory processes, primarily acute conditions such as sepsis, trauma, or acute respiratory distress where females tend to exhibit better prognosis compared with males. The mechanisms underlying these gender-dependent differences are multiple, probably involving hormonal and genetic factors, particularly the X chromosome. Although pH influences multiple immunological functions, gender differences in acid-base balance have been poorly investigated. In this review, we provide an update on gender differences in human susceptibility to inflammatory diseases. We additionally discuss the potential impact of acid-base balance on the gender bias of the inflammatory response in view of our recent observation that girls present higher neutrophilic inflammation and lower pH with a trend toward better prognosis in severe sepsis. We also highlight the potent role played by endothelial cells in gender differences of inflammation through activation of proton-sensing G protein-coupled receptors.

Prevalence, Risk Factors, and Serotype Distribution of Group B Streptococcus Colonization in HIV-Infected Pregnant Women Living in Belgium: A Prospective Cohort Study.

BACKGROUND: Group B streptococcus (GBS) infection is a leading cause of severe neonatal infection. Maternal GBS carriage during pregnancy is the main risk factor for both early-onset and late-onset GBS disease. High incidence of GBS infection has been reported in HIV-exposed but -uninfected infants (HEU). We aimed to determine the prevalence, characteristics, and risk factors for GBS colonization in HIV-infected and HIV-uninfected pregnant women living in Belgium. METHODS: Between January 1, 2011, and December 31, 2013, HIV-infected (n = 125) and -uninfected (n = 120) pregnant women had recto-vaginal swabs at 35-37 weeks of gestation and at delivery for GBS detection. Demographic, obstetrical, and HIV infection-related data were prospectively collected. GBS capsular serotyping was performed on a limited number of samples (33 from HIV-infected and 16 from HIV-uninfected pregnant women). RESULTS: There was no significant difference in the GBS colonization rate between HIV-infected and -uninfected pregnant women (29.6% vs 24.2%, respectively). HIV-infected women were more frequently colonized by serotype III (36.4% vs 12.5%), and the majority of serotype III strains belonged to the hypervirulent clone ST-17. Exclusively trivalent vaccine serotypes (Ia, Ib, and III) were found in 57.6% and 75% of HIV-infected and -uninfected women, respectively, whereas the hexavalent vaccine serotypes (Ia, Ib, II, III, IV, and V) were found in 97% and 100%, respectively. CONCLUSIONS: HIV-infected and -uninfected pregnant women living in Belgium have a similar GBS colonization rate. A trend to a higher colonization rate with serotype III was found in HIV-infected women, and those serotype III strains belong predominantly to the hypervirulent clone ST17.

Differential Susceptibility to Infectious Respiratory Diseases between Males and Females Linked to Sex-Specific Innate Immune Inflammatory Response.

It is widely acknowledged that males and females exhibit contrasting degrees of susceptibility to infectious and non-infectious inflammatory diseases. This is particularly observed in respiratory diseases where human males are more likely to be affected by infection-induced acute inflammations compared to females. The type and magnitude of the innate immune inflammatory response play a cardinal role in this sex bias. Animal models mimicking human respiratory diseases have been used to address the biological factors that could explain the distinct outcomes. In this review, we focus on our current knowledge about experimental studies investigating sex-specific differences in infection-induced respiratory diseases and we provide an update on the most important innate immune mechanisms that could explain sex bias of the inflammatory response. We also discuss whether conclusions drawn from animal studies could be relevant to human.

Increased Risk of Group B Streptococcus Invasive Infection in HIV-Exposed but Uninfected Infants: A Review of the Evidence and Possible Mechanisms.

Group B Streptococcus (GBS) is a major cause of neonatal sepsis and mortality worldwide. Studies from both developed and developing countries have shown that HIV-exposed but uninfected (HEU) infants are at increased risk of infectious morbidity, as compared to HIV-unexposed uninfected infants (HUU). A higher susceptibility to GBS infections has been reported in HEU infants, particularly late-onset diseases and more severe manifestations of GBS diseases. We review here the possible explanations for increased susceptibility to GBS infection. Maternal GBS colonization during pregnancy is a major risk factor for early-onset GBS invasive disease, but colonization rates are not higher in HIV-infected compared to HIV-uninfected pregnant women, while selective colonization with more virulent strains in HIV-infected women is suggested in some studies. Lower serotype-specific GBS maternal antibody transfer and quantitative and qualitative defects of innate immune responses in HEU infants may play a role in the increased risk of GBS invasive disease. The impact of maternal antiretroviral treatment and its consequences on immune activation in HEU newborns are important to study. Maternal immunization presents a promising intervention to reduce GBS burden in the growing HEU population.

The serum resistance-associated gene as a diagnostic tool for the detection of Trypanosoma brucei rhodesiense.

In the search for new diagnostic methods that would distinguish Trypanosoma brucei rhodesiense from T. b. brucei and T. b. gambiense, we have developed two polymerase chain reaction (PCR) primer sets. The first primer set was derived from the serum resistance-associated (SRA) gene of T. b. rhodesiense that confers resistance to lysis by normal human serum (NHS). The specificity of the SRA-based PCR was tested on 97 different trypanosome populations originating from various taxonomic groups, host species, and geographic regions. Only one of 25 T. b. rhodesiense samples was negative in this PCR, and none of 72 other samples were positive in this assay. Interestingly, a reference T. brucei strain (TREU927/4) currently used for genome sequencing was negative for the SRA gene; however, this strain was resistant to lysis by NHS. The second primer set was derived from a specific variant surface glycoprotein (VSG) expression site where the SRA gene is expressed (R-ES). This primer set identified the strain as T. b. rhodesiense in 17 of 17 SRA gene-positive strains in which it was tested. These data strongly suggest that expression of the SRA gene is generally involved in resistance to lysis by NHS in T. b. rhodesiense strains.

Delivery of biologically active anti-inflammatory cytokines IL-10 and IL-1ra in vivo by the Shigella type III secretion apparatus.

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF-alpha and IL-1beta mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.

CD40-CD40L interaction in immunity against protozoan infections.

Activation of the immune system against protozoan infections relies particularly on two specific signals provided by cognate interaction of T cells with antigen presenting cells (APCs). The first signal is attributed to binding of the T-cell receptor (TCR) to peptide/MHC complexes on the surface of APCs, whereas the second signal is triggered through binding of several costimulatory molecules on the surface of APCs with their corresponding receptors on T cells. Among these costimulatory signallings, CD40/CD40L interactions have been particularly investigated in protozoan infection models with regard to their potential to amplify cell-mediated immunity against intracellular parasites. This article reviews current studies of the potential role of CD40/CD40L interaction in the modulation of immune responses against some protozoan parasites and highlights recent developments regarding manipulation of this interaction for promoting control of parasite infections.

Cutting edge: small molecule CD40 ligand mimetics promote control of parasitemia and enhance T cells producing IFN-gamma during experimental Trypanosoma cruzi infection.

Host resistance to Trypanosoma cruzi infection depends on a type 1 response characterized by a strong production of IL-12 and IFN-gamma. Amplifying this response through CD40 triggering results in control of parasitemia. Two newly synthesized molecules (<3 kDa) mimicking trimeric CD40L (mini CD40Ls(-1) and (-2)) bind to CD40, activate murine dendritic cells, and elicit IL-12 production. Wild-type but not CD40 knockout mice exhibited a sharp decrease of parasitemia and mortality when inoculated with T. cruzi mixed with miniCD40Ls. Moreover, the immunosuppression induced by T. cruzi infection was impaired in mice treated with miniCD40Ls, as shown by proliferation of splenic lymphocytes, percentage of CD8(+) T cells, and IFN-gamma production. Mice surviving T. cruzi infection in the presence of miniCD40L(-1) were immunized against a challenge infection. Our results indicate that CD40L mimetics are effective in vivo and promote the control of T. cruzi infection by overcoming the immunosuppression usually induced by the parasites.

Transfection of Trypanosoma cruzi with host CD40 ligand results in improved control of parasite infection.

We have previously shown that infection by Trypanosoma cruzi, a parasitic protozoan, is reduced by injection of CD40 ligand (CD40L)-transfected 3T3 fibroblasts (D. Chaussabel, F. Jacobs, J. de Jonge, M. de Veerman, Y. Carlier, K. Thielemans, M. Goldman, and B. Vray, Infect. Immun. 67:1929-1934, 1999). This prompted us to transfect T. cruzi with the murine CD40L gene and to study the consequences of this transfection on the course of infection. For this, epimastigotes (Y strain) were electroporated with the pTEX vector alone or the pTEX-CD40L construct, and transfected cells were selected for their resistance to Geneticin G418. Then strain Y-, pTEX-, and pTEX-CD40L-transfected epimastigotes were transformed by metacyclogenesis into mammalian infective forms called Y, YpTEX, and YpTEX-CD40L trypomastigotes. Transfection of the CD40L gene and expression of the CD40L protein were assessed by reverse transcription-PCR and Western blot analysis. The three strains of parasites were infective in vitro for mouse peritoneal macrophages. When organisms were inoculated into mice, a very low level of parasitemia and no mortality were seen with the YpTEX-CD40L strain compared to the Y and YpTEX strains. Furthermore, the proliferative capacity and the secretion of gamma interferon were both preserved in spleen cells (SCs) from YpTEX-CD40L-infected mice but not with SCs from Y- and YpTEX-infected mice. These results suggest that the CD40L produced by transfected T. cruzi is involved in the modulation of an antiparasite immune response. Moreover, mice surviving YpTEX-CD40L infection resisted a challenge infection with the wild-type strain. Taken together, our data demonstrate the feasibility of generating a T. cruzi strain expressing a bioactive host costimulatory molecule that counteracts the immunodeficiency induced by the parasite during infection and enhances protective immunity against a challenge infection.

Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins.

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.