Associations Between Helicobacter pylori Infection,Co‐Morbid Infections,Gastrointestinal Symptoms,and Circulating Cytokines in African Children

Background: Refugee children have complex medical needs and often have multiple infections. The relationship between infection, gastrointestinal symptoms, and systemic inflammation is poorly understood. We investigated these parameters in refugee children with a high prevalence of Helicobacter pylori, helminth, and malaria infection. Materials and Methods: African refugee children were recruited at resettlement health screening. Data were collected on demography, gastrointestinal symptoms, co‐morbid infection, and serum for peripheral cytokine levels. Helicobacter pylori infection was diagnosed by a fecal‐based immunoassay. Results: Data from 163 children were analyzed, of which 84.0% were positive for H. pylori. Infected children were significantly older (9.2 years ± 3.7 vs 7.1 years ± 3.9, p = .01). Half the cohort (84/163, 51.5%) described gastrointestinal symptoms but these were not strongly associated with co‐morbid infections. Helicobacter pylori‐infected children had significantly lower circulating log‐interleukin‐8 (IL‐8) (odds ratio 0.61, 95% confidence interval (CI) 0.40, 0.94, p = .025). Helminth infections were common (75/163, 46%) and associated with elevated log‐IL‐5 (β: 0.42, 95% CI 0.077, 0.76). Children with malaria (15/163, 9.2%) had elevated log‐tumor necrosis factor‐α (TNFα) and log‐IL‐10 (β: 0.67, 95% CI 0.34, 1.0 and β: 1.3, 95% CI 0.67, 1.9, respectively). IL‐10 : IL‐12 ratios were increased in H. pylori‐infected children with malaria or helminth infections. Symptoms were generally not associated with levels of circulating peripheral cytokines irrespective of co‐morbid infection diagnosis. Conclusions: There is a high prevalence of asymptomatic H. pylori infection in recently resettled African refugee children. Gastrointestinal symptoms were not predictive of H. pylori nor of helminth infections. Serum cytokines, particularly IL‐5, IL‐10, and TNFα, were significantly elevated in children with malaria and helminth infections but not in those with H. pylori infection.     

Immunity to Salmonella from a dendritic point of view

Dendritic cells (DC) are the key link between innate and adaptive immunity. Features of DC, including their presence at sites of antigen entry, their ability to migrate from peripheral sites to secondary lymphoid organs, and their superior capacity to stimulate naïve T cells places them in this pivotal role in the immune system. DC also produce cytokines, particularly IL‐12, upon antigen encounter and can thus influence the ensuing adaptive immune response. As DC are phagocytic antigen‐presenting cells located at sites exposed to bacterial invaders, studies have been performed to gain insight into the role of DC in combating bacterial infections. Indeed, studies with Salmonella have shown that DC can internalize and process this bacterium for peptide presentation on MHC‐II as well as MHC‐I. DC can also act as bystander antigen‐­presenting cells by presenting Salmonella antigens after internalizing neighbouring cells that have undergone Salmonella‐induced apoptotic death. DC also produce IL‐12 and TNF‐α upon Salmonella encounter. Moreover, studies in a murine infection model have shown that splenic DC increase surface expression of co‐stimulatory molecules during infection, and DC contain intracellular bacteria. In addition, quantitative changes occur in splenic DC numbers in the early stages of oral Salmonella infection, and this is accompanied by redistribution of the defined DC subsets in the spleen of infected mice. DC from Salmonella‐infected mice also produce cytokines and can stimulate bacteria‐specific T cells upon ex vivo co‐culture. In addition, DC may play a role in the traversal of bacteria from the intestinal lumen. Studying the function of DC during Salmonella infection provides insight into the capacity of this sophisticated antigen‐presenting cell to initiate and modulate the immune response to bacteria.     

Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is the most common cause of bacterial, seafood‐related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro‐ and anti‐inflammatory cytokines by V. parahaemolyticus‐infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL‐1α, IL‐6, TNF‐α and IL‐10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T‐cell co‐stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.     

Early‐shared Mycobacterium bovis bacillus Calmette–Guérin sub‐strains induce Th1 cytokine production in vivo

Interleukin‐12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL‐2 and IFN‐γ. We have previously reported greater IL‐12 production from macrophages infected with early‐shared BCG sub‐strains (ex. BCG‐Japan, ‐Sweden) than from those infected with late‐shared BCG (ex. BCG‐Pasteur and ‐Connaught) 1 . In this study, we investigated the Th1 cytokine‐inducing activity of splenocytes co‐cultured with BCG‐infected DCs. Early‐shared BCG‐infected DCs produced IL‐12 and TNF‐α? Furthermore, when they were co‐cultured with purified protein derivative‐stimulated DCs, the splenocytes of mice immunized with BCG‐Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG‐Connaught. In conclusion, early‐shared BCG sub‐strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.

     

A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.     

Identification of lncRNA‐155 encoded by MIR155HG as a novel regulator of innate immunity against influenza A virus infection

Long noncoding RNAs (lncRNAs) are single‐stranded RNA molecules longer than 200 nt that regulate many cellular processes. MicroRNA 155 host gene (MIR155HG) encodes the microRNA (miR)‐155 that regulates various signalling pathways of innate and adaptive immune responses against viral infections. MIR155HG also encodes a lncRNA that we call lncRNA‐155. Here, we observed that expression of lncRNA‐155 was markedly upregulated during influenza A virus (IAV) infection both in vitro (several cell lines) and in vivo (mouse model). Interestingly, robust expression of lncRNA‐155 was also induced by infections with several other viruses. Disruption of lncRNA‐155 expression in A549 cells diminished the antiviral innate immunity against IAV. Furthermore, knockout of lncRNA‐155 in mice significantly increased IAV replication and virulence in the animals. In contrast, overexpression of lncRNA‐155 in human cells suppressed IAV replication, suggesting that lncRNA‐155 is involved in host antiviral innate immunity induced by IAV infection. Moreover, we found that lncRNA‐155 had a profound effect on expression of protein tyrosine phosphatase 1B (PTP1B) during the infection with IAV. Inhibition of PTP1B by lncRNA‐155 resulted in higher production of interferon‐beta (IFN‐β) and several critical interferon‐stimulated genes (ISGs). Together, these observations reveal that MIR155HG derived lncRNA‐155 can be induced by IAV, which modulates host innate immunity during the virus infection via regulation of PTP1B‐mediated interferon response.     

Quantification of the frequency and multiplicity of infection of respiratory- and lymph node-resident dendritic cells during influenza virus infection

Background

Previous studies have demonstrated that DC differentially regulate influenza A virus (IAV)–specific CD8 T cell responses in vivo during high and low dose IAV infections. Furthermore, in vitro infection of DC with IAV at low versus high multiplicities of infection (MOI) results in altered cytokine production and a reduced ability to prime naïve CD8 T cell responses. Flow cytometric detection of IAV proteins within DC, a commonly used method for detection of cellular IAV infection, does not distinguish between the direct infection of these cells or their uptake of viral proteins from dying epithelial cells.

Methods/Principal Findings

We have developed a novel, sensitive, single-cell RT-PCR–based approach to assess the infection of respiratory DC (rDC) and lymph node (LN)-resident DC (LNDC) following high and low dose IAV infections. Our results show that, while a fraction of both rDC and LNDC contain viral mRNA following IAV infection, there is little correlation between the percentage of rDC containing viral mRNA and the initial IAV inoculum dose. Instead, increasing IAV inoculums correlate with augmented rDC MOI.

Conclusion/Significance

Together, our results demonstrate a novel and sensitive method for the detection of direct IAV infection at the single-cell level and suggest that the previously described ability of DC to differentially regulate IAV-specific T cell responses during high and low dose IAV infections could relate to the MOI of rDC within the LN rather than the percentage of rDC infected.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Investigation of the immunomodulatory effects of Lactobacillus casei and Bifidobacterium lactis on Helicobacter pylori infection

Background: Lactobacillus and Bifidobacterium species have shown beneficial effects in the treatment of Helicobacter pylori infection; however, the mechanisms behind such effects are not fully understood. In this study, we have investigated the immunomodulatory effects of probiotics in a mouse model of H. pylori infection. Materials and methods: H. pylori‐infected C57BL/6 mice were treated with L. casei L26, B. lactis B94, or no probiotics for 5 weeks, respectively. Mice not infected with H. pylori were included as normal controls. Gastric histology, protein levels of interleukin (IL)‐1β, IL‐10, IL‐12/23p40, and H. pylori colonization density in the gastric tissues, as well as H. pylori‐specific antibodies were examined. Results: In mice receiving L. casei L26 and B. lactis B94, gastric neutrophil infiltration and IL‐1β were significantly decreased and IL‐10 was significantly increased as compared with mice receiving no probiotics. In mice receiving B. lactis B94, IL‐12/23p40 was significantly increased and H. pylori IgG was significantly reduced as compared with mice receiving no probiotics. No significant difference of H. pylori colonization was observed among the three groups of mice. Conclusion: The reduced level of IL‐1β and neutrophil infiltration observed in mice infected with H. pylori following treatment with L. casei L26 and B. lactis B94 resulted from a modulation of immune response rather than a decrease of H. pylori colonization. Furthermore, B. lactis B94 has the intrinsic ability to promote a Th1 immune response through an increase in IL‐12/IL‐23.     

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore‐forming toxin is detrimental for the host

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase‐1, which in turn triggers macrophage pyroptosis and IL‐1β/IL‐18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore‐forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase‐11 activation. Surprisingly, previous studies indicated that a T3SS‐induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS‐negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore‐forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll‐like receptors, and thus enhanced the expression of inflammatory proteins including pro‐IL‐1β and TNF‐α. However, mature‐IL‐1β and IL‐18 were undetectable in wild‐type mice, suggesting that ExlA failed to effectively activate caspase‐1. Nevertheless, caspase‐1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA‐induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome‐dependent process.     

Herpes simplex virus type 1 dysregulates anti‐fungal defenses preventing monocyte activation and downregulating toll‐like receptor‐2

We investigated the interplay occurring between pathogens in the course of dual infections, using an in vitro model in which the THP‐1 monocytic cell line is first infected with HSV‐1 and then exposed to Ca or Cn. These three pathogens share some pathogenic features: they cause opportunistic infections, target macrophages and are neurotropic. Here, we show that HSV‐1‐infected THP‐1 cells exhibited augmented phagocytosis against the two opportunistic fungi but reduced capability to counteract fungal infection: the better ingestion by monocytes was followed by facilitated fungal survival and replication. Reduced IL‐12 production was also observed. Cytofluorimetric analysis showed that HSV‐1‐infected monocytes exhibit: (i) downregulated TLR‐2 and TLR‐4, critical structures in fungal recognition; (ii) reduced expression of CD38 and CD69, known to be important markers of monocyte activation; and (iii) enhanced expression of apoptosis and necrosis markers, in the absence of altered cell proliferation. Overall, these findings imply that HSV‐1 infection prevents monocyte activation, thus leading to a significant dysfunction of the monocyte‐mediated anti‐Candida response; HSV‐1 induced apoptosis and necrosis of monocytes further contribute to this impairment.     

Cytokine and cellular responses to human herpesvirus‐6B in patients with B‐acute lymphoblastic leukemia

Primary infection with human herpesvirus‐6 (HHV‐6), is followed by its lifelong persistence in the host. Most T‐cell responses to HHV‐6 have been characterized using peripheral blood from healthy adults; however, the role of HHV‐6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV‐6 infection in patients with B‐acute lymphoblastic leukemia (B‐ALL) was analyzed. HHV‐6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti‐ and pro‐inflammatory cytokines (IL‐4, IL‐1, IL‐6, IL‐8, IL‐12p70, IL‐17a, TNF‐α and IFN‐γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV‐6 virus loads in blood. Characterization of T‐cell responses to HHV‐6 showed low specific T‐cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN‐γ‐producing T cells were detected in 0.03%–0.23% and in 0%–0.2% of CD4+T cells, respectively. Strong production of IL‐6 was detected in medium supernatants of challenged T‐cells whatever the HHV‐6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF‐α and IFN‐γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV‐6 in blood was identified, suggesting that HHV‐6 is not strongly associated with development of B‐ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV‐6B specific immune responses may be below the detection threshold of the assays used.     

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A (VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.     

Chlamydia pneumoniae induces interleukin‐6 and interleukin‐10 in human gingival fibroblasts

Chlamydia pneumoniae is an obligate intracellular Gram‐negative bacterium with a unique biphasic developmental cycle that can cause persistent infections. In humans, Chlamydia causes airway infection and has been implicated in chronic inflammatory diseases, such as asthma and atherosclerosis. In addition, recent studies demonstrated that patients with severe periodontitis can harbor C. pneumoniae, which can increase the risk for a host inflammatory response with weighty clinical sequelae. Previous studies have established that periodontal pathogenic bacteria (i.e. Gram‐negative bacteria) can induce the synthesis and release of cytokines and other inflammatory mediators in human gingival fibroblasts. HGF are resident cells of the periodontium that respond to receptor stimulation by producing a variety of substances including cytokines and growth factors. Our results demonstrate that after 48 hr of incubation with viable C. pneumoniae HGF showed a proliferative response, as seen by both colorimetric MTT assay and direct cell count (30% and 35%, respectively). In addition, HGF incubated with viable or UV light‐inactivated C. pneumoniae organisms showed an increase in the levels of IL‐6 and IL‐10, but not IL‐4; on the contrary, HGF infected with heat‐killed bacteria did not show a significant production of any of the cytokines considered. In conclusion, the present study suggests that C. pneumoniae may modulate the expression of IL‐6 and IL‐10 by human gingival fibroblasts. Further studies are warranted to clarify the molecular mechanisms of C. pneumoniae in the regulation of cytokine expression by host cells and to elaborate the relevant clinical implications.     

Site‐specific and endothelial‐mediated dysfunction of the alveolar‐capillary barrier in response to lipopolysaccharides

Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar‐capillary barrier (ACB) in the lung. In this study, we analyse the site‐specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co‐cultures of H441 with endothelial cells cultured on Transwells^® were treated with LPS from the apical or basolateral compartment. Barrier properties were analysed by the transepithelial electrical resistance (TEER), by transport assays, and immunostaining and assessment of tight junctional molecules at protein level. Furthermore, pro‐inflammatory cytokines and immune‐modulatory molecules were evaluated by ELISA and semiquantitative real‐time PCR. Liquid chromatography–mass spectrometry‐based proteomics (LS‐MS) was used to identify proteins and effector molecules secreted by endothelial cells in response to LPS. In co‐cultures treated with LPS from the basolateral compartment, we noticed a significant reduction of TEER, increased permeability and induction of pro‐inflammatory cytokines. Conversely, apical treatment did not affect the barrier. No changes were noticed in H441 monoculture upon LPS treatment. However, LPS resulted in an increased expression of pro‐inflammatory cytokines such as IL‐6 in OEC and in turn induced the reduction of TEER and an increase in SP‐A expression in H441 monoculture, and H441/OEC co‐cultures after LPS treatment from basolateral compartment. LS‐MS‐based proteomics revealed factors associated with LPS‐mediated lung injury such as ICAM‐1, VCAM‐1, Angiopoietin 2, complement factors and cathepsin S, emphasizing the role of epithelial–endothelial crosstalk in the ACB in ALI/ARDS.     

Babesia: the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice

In the present study, we investigated the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice. Pre-treatment with "EqStim" (a commercially available immunostimulant containing killed P. acnes) showed significant resistance to both infections. To elucidate the immunological status in the mice, the concentrations of multiple cytokines were measured in serum collected from infected mice. After B. microti infection, the levels of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12p70, and tumor necrosis factor (TNF)-alpha in the treated group were significantly lower than in the control group. In contrast, after B. rodhaini infection, only IL-12p70 and TNF-alpha were detectable at significantly higher levels in the treated group than in the control group. The present findings indicated the protective effects of killed P. acnes on rodent babesiosis even with different immune responses between the B. microti and B. rodhaini infections. Killed P. acnes might be a powerful tool for the control of serious livestock babesiosis.