Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1β and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.     

The expression analysis of inflammatory and antimicrobial genes in the goldfish (Carassius auratus L.) infected with Trypanosoma carassii

We report results of a comprehensive analysis of inflammatory gene expression during the course of infection of Trypanosoma carassii in the goldfish. We observed significant increases in mRNA levels of genes encoding pro-inflammatory cytokines IFN-γ, TNFα1 and TNFα2; IL-1β-1 and IL-1β-2; IL-12-p35 and IL-12-p40; CCL1; CXCL8, anti-inflammatory cytokines IL-10 and TGFβ and iNOS A and iNOS B, using quantitative PCR. Expression levels and profiles of these cytokines and iNOS isoforms varied in the different tissues (kidney, spleen, liver) of goldfish during the course of T.?carassii infection. The expression of majority of genes that encode pro- and anti-inflammatory cytokines were up-regulated during the acute phase of infection (days 7-21 post-infection). The mRNA levels of these cytokines returned to normal levels or were down-regulated during the elimination phase of infection (days 28-56), with exception of IL-10 in the spleen and liver of infected fish. A parallel up-regulation of IFN-γ and IL-10 mRNA levels were observed in all tissues of infected fish during the acute phase of the infection. The expression of iNOS genes (iNOS A and B) was significantly delayed (day 14?pi) in the kidney, liver and spleen of infected fish. These results provide insights into the interaction between T.?carassii and goldfish, and suggest that Th1/Th2-like responses may be important for controlling T.?carassii infection in the goldfish.     

Fate of Francisella noatunensis, a pathogen of Atlantic cod Gadus morhua, in blue mussels Mytilus edulis

Francisellosis, caused by the bacterium Francisella noatunensis, is one of the most severe diseases affecting farmed cod, and has caused great economic loss for the cod farming industry in Norway. We studied the fate of F. noatunensis in the marine environment, focusing on the role of blue mussels. In experimental challenges, waterborne F. noatunensis was rapidly filtered by the blue mussel and transported to the digestive diverticulae. The bacteria passed through the entire digestive system. Intraperitoneal injection of cod with suspensions prepared from faeces collected from challenged mussels resulted in the development of francisellosis in the recipients, demonstrating that some bacteria were alive and infective when shed in mussel faeces. Bacterial clearance from the mussels was relatively fast, and no evidence was found, suggesting that the bacterium is capable of persisting or multiplying in the mussel tissues. A cohabitation experiment with cod and mussels previously exposed to F. noatunensis did not lead to infection in cod. A direct transmission from contaminated mussels to cod was thus not demonstrated; however, faeces particles with infective bacteria may play a role in the transmission of the bacterium in marine food chains.     

Molecular characterisation and expression analysis of interferon gamma in Atlantic cod (Gadus morhua)

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. In this study, the cDNA and genomic sequences of Atlantic cod IFN-γ were cloned and found to encode a putative protein containing 194 amino acids with a 24 amino acid signal peptide sequence. The gene is composed of four exons and three introns similar to IFN-γ genes of other vertebrates. The cod IFN-γ showed only 14–29% amino acid identity with other fish IFN-γ and 9–17% identity with IFN-γ from higher vertebrates. However, cod IFN-γ possesses the typical IFN-γ motifs in the C-terminal end of the protein and displays an alpha-helix structure similar to mammalian IFN-γ. The promoter region contains a putative ISRE element indicating up-regulation by type I IFNs and dsRNA. Real time RT-PCR analysis confirmed that IFN-γ gene expression was up-regulated in organs of cod injected with the dsRNA polyinosinic:polycytidylic acid (poly I:C), which is a strong inducer of type I IFNs. Injection of cod with formalin-killed Vibrio anguillarum also increased IFN-γ expression in head kidney, but to a much lesser extent than poly I:C. The gene expression results thus indicate a role for IFN-γ in innate immune response against both virus and bacteria in Atlantic cod.     

IFN-β mediates suppression of IL-12p40 in human dendritic cells following infection with virulent Francisella tularensis

Active suppression of inflammation is a strategy used by many viral and bacterial pathogens, including virulent strains of the bacterium Francisella tularensis, to enable colonization and infection in susceptible hosts. In this study, we demonstrated that virulent F. tularensis strain SchuS4 selectively inhibits production of IL-12p40 in primary human cells via induction of IFN-β. In contrast to the attenuated live vaccine strain, infection of human dendritic cells with virulent SchuS4 failed to induce production of many cytokines associated with inflammation (e.g., TNF-α and IL-12p40). Furthermore, SchuS4 actively suppressed secretion of these cytokines. Assessment of changes in the expression of host genes associated with suppression of inflammatory responses revealed that SchuS4, but not live vaccine strain, induced IFN-β following infection of human dendritic cells. Phagocytosis of SchuS4 and endosomal acidification were required for induction of IFN-β. Further, using a defined mutant of SchuS4, we demonstrated that the presence of bacteria in the cytosol was required, but not sufficient, for induction of IFN-β. Surprisingly, unlike previous reports, induction of IFN-β by F. tularensis was not required for activation of the inflammasome, was not associated with exacerbation of inflammatory responses, and did not control SchuS4 replication when added exogenously. Rather, IFN-β selectively suppressed the ability of SchuS4-infected dendritic cells to produce IL-12p40. Together, these data demonstrated a novel mechanism by which virulent bacteria, in contrast to attenuated strains, modulate human cells to cause disease.     

Astragalus polysaccharides enhance the humoral and cellular immune responses of hepatitis B surface antigen vaccination through inhibiting the expression of transforming growth factor β and the frequency of regulatory T cells

Astragalus polysaccharides (APS), extracted from the root of Astragalus membranaceus, a traditional Chinese medicinal herb, have extensive pharmacological and strong immunomodulatory effects. In this study, the potential adjuvant effect of APS on humoral and cellular immune responses to hepatitis B subunit vaccine was investigated. Coadministration of APS with recombinant hepatitis B surface antigen significantly increased antigen-specific antibody production, T-cell proliferation and CTL (cytotoxic T lymphocyte) activity. Production of interferon-γ (IFN-γ), interleukin-2 (IL-2) and IL-4 in CD4(+) T cells and of IFN-γ in CD8(+) T cells were dramatically increased. Furthermore, expression of the genes PFP, GraB, Fas L and Fas were up-regulated; interestingly, expression of transforming growth factor β (TGF-β) and the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) were down-regulated. Expression of Toll-like receptor 4 (TLR4) was significantly increased by administration of APS. Together, these results suggest that APS is a potent adjuvant for the hepatitis B subunit vaccine and can enhance both humoral and cellular immune responses via activating the TLR4 signaling pathway and inhibit the expression of TGF-β and frequency of Treg cells.     

A critical role for SOCS3 in innate resistance to Toxoplasma gondii

The innate and adaptive immune responses that confer resistance to the intracellular pathogen Toxoplasma gondii critically depend on IL-12 production, which drives interferon-γ (IFN-γ) expression. Certain cytokines can activate STAT3 and limit IL-12 production to prevent infection-associated immune pathology, but T.?gondii also directly activates STAT3 to evade host immunity. We show that suppressor of cytokine signaling molecule 3 (SOCS3), a target of STAT3 that limits signaling by the pleiotropic cytokine IL-6, is upregulated in response to?infection but is dispensable for the immune-inhibitory effects of T.?gondii. Unexpectedly, mice with targeted deletion of SOCS3 in macrophages and neutrophils have reduced IL-12 responses and succumb to toxoplasmosis. Anti-IL-6 administration or IL-12 treatment blocked disease susceptibility, suggesting that in the absence of SOCS3, macrophages are hypersensitive to the anti-inflammatory properties of IL-6. Thus, SOCS3 has a critical role in suppressing IL-6 signals and promoting immune responses to control T.?gondii infection.     

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.     

Functional role of endogenous CD14 in lipopolysaccharide-stimulated bone resorption

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions, and one of these roles is that of a powerful stimulator of bone resorption. However, the mechanism by which LPS stimulates bone resorption is not yet understood. In the present study, we show, by using mouse embryonic calvarial cells, that endogenous CD14 and interleukin-1β (IL-1β) play an important role in the LPS-mediated bone resorption and that interferon-γ (IFN-γ)functions as a strong inhibitor of this resorption by suppressing LPS-stimulated expression of CD14 and IL-1β genes in the calvarial cells. We observed that LPS-stimulated differentiation of osteoclastic cells and bone and resorption were markedly neutralized by anti-mouse CD14 antibody were clearly inhibited by anti-sense CD14 oligonucleotide treatment. In addition, because LPS stimulated CD14 gene expression in the calvarial cells, these observations demonstrate the precise role of endogenous CD14 in LPS-stimulated differentiation of osteoclastic cells and boneresorption. However, the stimulation of the differentiation of osteoclastic cells and bone resorption was also inhibited by anti-mouse IL-1β antibody. Interestingly, anti-sense CD14 oligonucleotide inhibited LPS-stimulated expression of the IL-1β gene in the calvarial cells. These observations suggest a functional role of endogenous CD14 in LPS-stimulated expression of the IL-1β gene in the cells. Because IFN-γ is a potent inhibitor of bone resorption stimulated by IL-1, in additional experiments, we examined whether IFN-γ is able to inhibit LPS-stimulated differentiation of osteoclastic cells and bone resorption. We found that IFN-γ inhibited these stimulations by suppressing CD14 and IL-1β genes in the calvarial cells. The present study thus clearly demonstrates a functional role of endogenous CD14 in LPS-stimulated bone resorption. J. Cell. Physiol. 173:301–309, 1997. © 1997 Wiley-Liss, Inc.     

New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy

Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate-induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1β, and IL-1β in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1β, and IL-1β can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.     

Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.     

IL-6 inhibits IFN-γ induced autophagy in Mycobacterium tuberculosis H37Rv infected macrophages

The significance of IL-6 production in tuberculosis is yet to be fully elucidated, although it is known for quite some time that IL-6 interferes with IFN-γ induced signal. In order to know which cellular process induced by IFN-γ is actually counteracted by IL-6, we studied the role of IL-6 on IFN-γ induced autophagy formation in virulent Mycobacterium tuberculosis infection in THP-1 cells, since it is well characterized that induction of autophagy by IFN-γ eliminates intracellular mycobacterium by overcoming the phagosome maturation block imposed by bacilli. We report here that IL-6 inhibits both IFN-γ and starvation induced autophagy in M. tuberculosis H37Rv infected cells. M. tuberculosis H37Rv infection results in time dependent production of IL-6 in THP-1 cells and neutralization of this endogenous IL-6 by anti-IL-6 antibody significantly enhances the IFN-γ mediated killing of the intracellular bacteria. IL-6 time dependently lowers Atg12-Atg5 complex and therefore inhibits autophagosome biogenesis rather than autophagolysosome formation. IL-6 also affects IFN-γ mediated stimulation of mTOR, p-38 and JNK pathways. These results clearly indicate that virulent mycobacteria strategically upregulate IL-6 production to combat innate immunity.     

IL-11 expression in retinal and corneal cells is regulated by interferon-γ

Interleukin-11 (IL-11) is an anti-apoptotic, anti-inflammatory cytokine with hematopoietic potential. The expression and protective actions of IL-11 have not been explored in the eye. The expression of IL-11 in primary cultures of human retinal pigment epithelial (HRPE) and human corneal fibroblast (HCRF) cells were evaluated in these studies. Constitutive secretion of IL-11 was not observed in either HRPE or HCRF. TNF-α + IL-1 induced IL-11 secretion and this production was inhibited by NFκB pathway inhibitors. IFN-γ significantly inhibited TNF-α and IL-1 induced IL-11 secretion and inhibitors of JAK-STAT pathway reversed this inhibition. TGF-β induced IL-11 secretion that was blocked by TGF-β receptor 1 inhibitor but not by IFN-γ. RT-PCR analysis confirmed the effects of IL-1, TNF-α, IFN-γ and TGF-β on IL-11 secretion at mRNA levels. Our results demonstrate that IL-11 is dramatically up regulated in retina and cornea cells and that IFN-γ is a physiological inhibitor of IL-11 expression.     

益生菌对感染小鼠早期IFN-γIL-4的影响

目的研究口服益生菌对鼠伤寒沙门菌（STM）感染小鼠Th细胞因子的影响,以探讨益生菌抗沙门菌感染的免疫学机制。方法将95只Balb／c小鼠分为4组,分别为益生菌组（P）、益生菌对照组（Pc）、正常感染组（I）和对照组（C）。P组口服益生菌,I组口服生理盐水,均予等剂量STM口服感染,Pc组接种益生菌但不感染STM,C组不作任何处理。各组动物在11个不同时点处死,观察小肠、肝脏、脾脏病理改变,ELISA测量血清IFN-γ、IL-4表达。结果益生菌组器官组织的病理改变轻微,IFN-γ较正常感染组明显增加（Xp-1=66．52,P=0．001）,且其表达在感染最初的1h和后期的72h分别出现两个高峰;IL-4明显降低（Xp-1=-29．02,P〈0．001）,且较稳定。IFN-γ／IL4比值扩大（Xp-1=2．64,P〈0．001）。结论口服益生菌使小鼠保持有利于抗STM感染的Th1型反应,减轻了STM对机体的免疫损伤。     

Francisella noatunensis subsp. noatunensis is the aetiological agent of visceral granulomatosis in wild Atlantic cod Gadus morhua

During the 1980s and 1990s wild-caught cod displaying visceral granulomatosis were sporadically identified from the southern North Sea. Presumptive diagnoses at the time included mycobacterial infection, although mycobacteria were never cultivated or observed histologically from these fish. Farmed cod in Norway displaying gross pathology similar to that identified previously in cod from the southern North Sea were recently discovered to be infected with the bacterium Francisella noatunensis subsp, noatunensis. Archived formalin-fixed paraffin-embedded tissues from the original North Sea cases were investigated for the presence of Mycobacterium spp. and Francisella spp. using real-time polymerase chain reaction, DNA sequencing and immunohistochemistry. Whilst no evidence of mycobacterial infection was found, F. noatunensis subsp. noatunensis was identified in association with pathological changes consistent with Francisella infections described from farmed cod in recent years. This study shows that francisellosis occurred in wild-caught cod in the southern North Sea in the 1980s and 1990s and demonstrates that this disease predates intensive aquaculture of cod.     

The biological basis of severe outcomes in Anaplasma phagocytophilum infection

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an acute disease in humans that is also often subclinical. However, 36% are hospitalized, 7% need intensive care, and the case fatality rate is 0.6%. The biological basis for severe disease is not understood. Despite A.?phagocytophilum's mechanisms to subvert neutrophil antimicrobial responses, whether these mechanisms lead to disease is unclear. In animals, inflammatory lesions track with IFNγ and IL-10 expression and infection of Ifng(-/-) mice leads to increased pathogen load but inhibition of inflammation. Suppression of STAT signaling in horses impacts IL-10 and IFN-γ expression, and also suppresses disease severity. Similar inhibition of inflammation with infection of NKT-deficient mice suggests that innate immune responses are key for disease. With severe disease, tissues can demonstrate hemophagocytosis, and measures of macrophage activation/hemophagocytic syndromes (MAS/HPS) support the concept of human granulocytic anaplasmosis as an immunopathologic disease. MAS/HPS are related to defective cytotoxic lymphocytes that ordinarily diminish inflammation. Pilot studies in mice show cytotoxic lymphocyte activation with A.?phagocytophilum infection, yet suppression of cytotoxic responses from both NKT and CD8 cells, consistent with the development of MAS/HPS. Whether severity relates to microbial factors or genetically determined diversity in human immune and inflammatory response needs more investigation.