TNF receptor/ligand superfamily members signal through pathways giving rise to proteins that regulate lymphocyte proliferation, activation, differentiation and survival. Absence of TNF ligands OX40L and CD30L impairs survival of GC T cells and affinity maturation of antibody responses. Direct effects of these molecules on B cells in antibody responses are not characterised. I dissected roles of OX40 and CD30 for B cells using T-independent type II (TI-II) antigen NP-Ficoll. Humoral immunity is impaired in OX40 deficiency. Defects in class switched and non-class switched antibody production are due to reduced development of antigen-specific switched and non-switched plasma cells. CD30 has an opposing role, deficiency results in similar or higher switched and non-switched antibody titres and higher numbers of antigen-specific plasma cells that develop rapidly. This may explain why in OX40/CD30 double deficiency, there is a less pronounced defect than in OX40 single deficiency. B cell intrinsic roles are revealed for OX40 and CD30 that suggest OX40 on B cells is critical for TI-II plasmablast differentiation or survival and B cell CD30 inhibits onset of plasmablast differentiation.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser. Karolinska International Research Training Program (KIRT).

Innate immunity is a branch of the immune system that is responsible for controlling the early events of pathogen infection. One of the key components of the innate immune systems arsenal are the interferon (IFN) cytokines. IFNs are small signalling proteins that are released by cells in response to invading pathogens, and viruses in particular. They are named for their ability to interfere with viral replication. The result of IFN signalling is the up-regulation of a diverse collection of genes termed interferon-stimulated genes (ISGs). These genes act in synchrony to limit the replication of viruses. The protein products of ISGs are involved in a multitude of cellular pathways that limit replication and additionally intercept viral proteins and nucleic acid directly. Some of these ISGs are mediators of an important cell-death response, apoptosis. Apoptosis is a vital component of innate immune signalling and controls viral replication by sacrificing the infected cell to limit further infection of neighbouring cells. The function of specific ISGs in mediating this response is poorly understood.

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No description available.

Introduction Autophagy is a cellular process whereby elements within cytoplasm become engulfed within membrane vesicles and trafficked to fuse with lysosomes. This is a common cellular response to starvation, allowing non-essential cytoplasmic contents to be recycled in times of energy deprivation. However, autophagy also plays an important role in immunity and inflammation, where it promotes host defence and down-regulates inflammation. A specialised bacterial virulence mechanism, the type III secretion system (T3SS) in Pseudomonas aeruginosa (PA), an extracellular bacterium, is responsible for the activation of the inflammasome and IL-1β production, a key cytokine in host defence. The relationship between inflammasome activation and induction of autophagy is not clear. Hypothesis and aims The central hypothesis is that induction of autophagy occurs following PA infection and that this process will influence inflammasome activation in macrophages. Our aims were to determine the role of the T3SS in the induction of autophagy in macrophages following infection with PA, and to investigate the effects of autophagy on inflammasome activation and other pro-inflammatory pathways following infection with these bacteria. Materials and methods Primary mouse bone marrow macrophages BMDMs were infected with PA, in vitro. Induction of autophagy was determined using five different methods: - electron microscopy, immunostaining of the autophagocytic marker LC3, FACS, RT-PCR assays for autophagy genes, and post-translational conjugation of phosphatidylethanoloamine (PE) to LC3 using Western blot. Inflammasome activation was measured by secretion of active IL-1β and caspase-1 using ELISA and Western blot. Functional requirements of proteins were determined using knockout animals or SiRNA mediated knockdown. Result and Conclusions PA induced autophagy that was not dependent on a functional T3SS but was dependent on TLR4 and the signaling molecule TRIF. PA infection also strongly induced activation of the inflammasome which was absolutely dependent on a functional T3SS. We found that inhibition of inflammasome activation increased autophagy, suggesting that the inflammasome normally inhibits this process. Further experiments showed that this inhibitory effect was due to the proteolytic action of caspase-1 on the signaling molecule TRIF. Using a construct of TRIF with a mutation in the proteolytic cleavage site, prevented caspase-1 cleavage and increased autophagy. TRIF is also involved in the production of interferon-β following infection. We also found that caspase-1 cleavage of TRIF down-regulated this pathway as well. Caspase-1 mediated inhibition of TRIF-mediated signaling is a novel pathway in the inflammatory response to infection. It is potentially amenable to therapeutic intervention. Recognition of a pathogen infection is a key function of the innate immune system that allows an appropriate defensive response to be initiated. One of the most important innate immune defences is provided by a multi-subunit cytoplasmic platform termed the inflammasome that results in production of the cytokine IL-1β. The human pathogen Pseudomonas aeruginosa activates the inflammasome following infection in a process that is dependent on a specialized bacterial virulence apparatus, the type III secretory system (T3SS). Here, we report the novel finding that this infection results in mitochondrial damage and release of mitochondrial DNA into the cytoplasm. This initiates activation of an inflammasome based on the protein NLRC4. Autophagy induced during infection removes damaged mitochondria and acts to down-regulate NLRC4 activation following infection. Our results highlight a new pathway in innate immune activation following infection with a pathogenic bacterium that could be exploited to improve outcomes following infection.

Much is known about the disease pathology related to schizophrenia, however, little is known with regards to its aetiology. Recent evidences suggest a neurodevelopmental hypothesis for schizophrenia where environmental factors including: infection, stress and malnutrition, can adversely affect the pregnant mother thereby elevating the risk for schizophrenia developing in the offspring during adulthood (Meyer et al., 2008d; Meyer and Feldon, 2009; 2012; Forrest et al., 2012; Meyer, 2013). Since a variety of viral and bacterial infections in animal models have demonstrated to increase the risk in schizophrenia, it is proposed that factors common to the immune response may mediate this link. While many laboratories have reported several behavioural abnormalities following maternal immune activation, we sought to examine molecular changes following poly(I:C) exposure, a synthetic viral mimetic, in the pregnant mother and assessed a range of protein markers with known developmental roles, since an appreciable understanding of the molecular alterations taking place would permit suitable therapies to follow. Interestingly, poly(I:C) was able to induce a range of changes resembling those observed during schizophrenia, where the major NMDA receptor subunit GluN1 and α-Synuclein was reduced in postnatal day 21 animals born to mothers treated with poly(I:C) during gestation days 14, 16 and 18. Furthermore, these changes suggest a mechanism by which maternal immune activation may lead to the subsequent emergence of schizophrenia. Another aspect of this work examined the role of the kynurenine pathway on brain development. There is increasing evidence suggesting the involvement of the kynurenine pathway, a biochemical pathway responsible for the oxidative metabolism of tryptophan, in the disease pathology of schizophrenia, including neurodegenerative disorders such as Parkinson’s, Alzheimer’s and Huntington’s disease (Giorgini et al., 2005; Ting et al., 2009; Bonda et al., 2010). Since immune activation induces the activation of the kynurenine pathway, it was hypothesised that alterations in central kynurenine concentrations during development may be involved in mediating the subsequent increased risk for schizophrenia (Forrest et al., 2013, Khalil et al., 2013, 2014). As very little is known about the physiological activity of the kynurenine pathway during development, we sought to examine the potential consequence of disrupting this pathway and examining its effects upon brain development. Therefore, a kynurenine monooxygenase inhibitor, Ro61-8048, was administered to pregnant rats during gestation day 14, 16, and 18, that would inhibit the synthesis of the neurotoxic metabolite quinolinic acid, while redirecting the pathway to increase the neuroprotectant kynurenic acid. Brain development was assessed by examining changes in protein expression of markers intimately involved in synaptic transmitter release machinery, neurogenesis and many aspects of neuronal development. Interestingly, we found the kynurenine pathway is highly active during brain development, and induces a variety of changes in protein markers that may be involved in precipitating a range of neuronal and cognitive deficits. While Ro61-8048 induced no changes in the embryo brains at 5 and 24 h following treatment, delayed changes were seen in postnatal day 21 animals displaying a decrease in RhoB expression as examined in the western blots. Since the full blow symptoms of schizophrenia become apparent during early adulthood, we sought to examine any changes in protein expression in postnatal day 60 animals in regions of the cortex, hippocampus, midbrain and cerebellum. Interestingly, profound alterations were seen in doublecortin and the netrin receptors responsible for axonal guidance. Perhaps the most striking protein change in the postnatal day 60 animals is the significant alteration induced in the expression of disrupted in schizophrenia (DISC)-1, a protein strongly linked with schizophrenia. Glutamate function was assessed as indicated by the density of glutamate transporters, VGLUT-1 and VGLUT-2, in the CA1 region of the hippocampus of postnatal day 60 animals using immunocytochemistry. While the relative density of glutamate transporters were substantially increased, there were no changes in the GABA transporters, indicating that while GABA transmission remained the same, glutamate function may have increased in the absence of an increase in synaptic connections. Spine densities of pyramidal neurons in the hippocampus were also examined, using the golgi-impregnation method, to reveal a significant loss in spines of the apical and basal dendrites, consistent with reports in schizophrenia. To conclude, the kynurenine pathway is highly active during development, and alterations in central kynurenines during pregnancy, as induced by environmental factors such as stress and infection, may be involved in the subsequent emergence of neurodevelopmental disorders.

Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress. No specific treatment methods are available and little is known about its aetiology. The aims of this study were:- 1) to identify the bacterial flora, including uncultivable and potentially novel species, in healthy cats and those with FCGS, using 16S rRNA gene sequencing in combination with conventional culture methods; 2) to investigate the viral status of cats with and without FCGS; 3) to assess the immune response by investigating the expression of cytokine and Toll-like receptor (TLR) genes in tissue biopsies from normal cats and those with FCGS; 4) to investigate the histopathological changes in tissue biopsies from normal cats and those with FCGS, 5) to assess putative risk factors for FCGS by the use of a questionnaire-based study. Oral swabs, mucosal biopsies and blood were collected and the location of the oral lesions was recorded. A total of 32 cats with FCGS and 16 normal cats were included in the study. Bacteria were identified from swabs by use of 16S rRNA gene sequencing and by conventional culture methods. Blood samples and swabs were used for diagnosis of infection with feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), feline herpes virus 1 (FHV-1), feline calicivirus (FCV) and for blood biochemistry and haematology. Gene expression levels for TLR2, TLR3, TLR4, TLR7 and TLR9, and cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α and IFN-γ mRNA were determined using quantitative PCR in biopsy samples from healthy cats and cats with FCGS. Histopathological examination of the tissue biopsies was done using hematoxylin and eosin (H&E) staining. In the healthy group, 16S rRNA gene sequencing demonstrated that the most prevalent bacteria were part of the Proteobacteria and Bacteroidetes phyla, plus a group of uncultured bacteria. The most prevalent species in the healthy group were Xanthomonadaceae bacterium (6.2 % of clones analysed), Capnocytophaga canimorsus (5.4%), Capnocytophaga cynodegmi (4.8%), Bergeyella species (4.5%) and Pasteurella multocida subspecies septica (4.4%). Uncultured bacteria accounted for 29% of the clones analysed. In the FCGS group most of the identified species were part of the phylum Proteobacteria. The most prevalent species in the FCGS group were P. multocida subsp. multocida (14.1%) P. multocida subsp. septica (11.5%), Pseudomonas sp. (7.3%), Tannerella forsythia (6.6%) and Porphyromonas circumdentaria (5.6%). A variety of uncultured bacteria represented 7.7% of all analysed FCGS clones. The culture data showed the most prevalent bacteria in the healthy group were P. multocida subsp. septica (9.9%), and uncultured bacteria (30.5%). In the FCGS group the most prevalent isolates were P. multocida subsp septica and P. multocida subsp. multocida (both 9.9%). Uncultured bacteria accounted for 21.7% of all isolates. FCV was detected in 71% of cats with FCGS and in 13.3% of normal cats. FeLV antigen was detected in 33.3% of normal cats but not in any cats with FCGS. FIV antibodies were detected in 3.4% of cats with FCGS and in 33.3% of normal cats. FHV-1 was detected in 6.9% of cats with FCGS, but was not detected any of the normal cats. In the FCGS group a significant increase was seen in the expression of TLR2 and TLR7 genes as well as TNF-α, IFN-γ, IL-1β and IL-6 cytokine genes. The healthy cats and cats with FCGS in the study that were found to harbour T. forsythia and P. circumdentaria showed an increase in the expression of several TLR and cytokine genes when compared to the group of cats in which these bacterial species were absent. The most severely inflamed sites in the oral cavity of cats with FCGS included the tissue lateral to the palatoglossal folds and the maxillary attached gingiva. Histopathological analysis of the tissue from the palatoglossal folds showed two types of infiltrates:- 1) a combination of lymphocytes and plasma cells, most often seen in the milder inflamed tissue samples; 2) a predominantly plasmacytic infiltration, most often seen in the severely inflamed tissue samples. Preliminary data from a questionnaire-based epidemiological study showed that the presence of potential environmental stress factors such as no ability to roam outdoors and the presence of more than one cat in the household is significantly higher in cats with FCGS when compared to normal cats. This study highlights the possibility of a multifactorial aetiology for FCGS in which FCV, specific bacteria and stress factors may play an important role. Although species from the Bacteroidetes phylum appeared to be capable of eliciting an immune response, these were not the most prevalent species in the FCGS group. A shift could be seen in the composition of the bacterial flora when healthy cats and those with FCGS were compared.

Despite advances in treatment, peritoneal dialysis (PD)-associated peritonitis remains a major cause of morbidity and mortality in PD patients. Given that peritonitis can be the proximate cause of technique failure and cause ultrafiltration failure at a later time, it is important to understand the peritoneal immune response, microbiology and outcomes of these infections. Data presented in this thesis have shown that leukocytes are recruited to the peritoneal cavity, starting with a rapid accumulation of neutrophils, which are later replaced by a population of mononuclear cells, including monocytes/macrophages and T cells during acute peritonitis. Of note, Vγ9/Vδ2 T cells are also recruited to the peritoneal cavity in the early stage, which implies a significant role of Vγ9/Vδ2 T cells as early responders in acute peritonitis. In patients with acute peritonitis, the capacity of the causative pathogen to produce (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), together with the infiltration of activated Vγ9/Vδ2 T cells are important risk factors and possible predictors of patient outcomes from infection. By performing a detailed immunological and microbiological analysis in PD patients on the first day of peritonitis, our findings provide proof of concept that acute bacterial infections indeed leave characteristic disease-specific ‘immune fingerprints’ of diagnostic and prognostic value. Local fingerprints not only discriminated between episodes of culture-negative and culture-positive PD-associated peritonitis but also predicted infections caused by Gram− or Gram+ bacteria. HPMC play an important role in maintaining homeostasis of the peritoneal immunity. Our data revealed the regulation of Vγ9/Vδ2 T cells by HPMC and demonstrated that resting HPMC were potent suppressors of Vγ9/Vδ2 T-cell cytokine production and proliferation in the presence of HMB-PP. Collectively, these findings improve our insight into the complex cellular interactions in PD-associated peritonitis and peritoneal homeostasis, identify novel biomarkers of possible diagnostic and predictive value and highlight new avenues for therapeutic intervention.

Introduction: Varying degrees of periodontal disease affect the majority of the population. Severe forms of periodontitis have a considerable impact on oral health and quality of life. Periodontitis results from imbalances in the oral microbiome and the host immune response. The mainstay of periodontal treatment – removal of dental plaque – is only partially successful. B cells infiltrate the gingiva of periodontitis patients, but their role in pathology has not been well characterised. The overarching aim of this research was to better characterise the role of B cells in periodontitis. Periodontitis shares similarities in risk factors and aspects of immunopathology with rheumatoid arthritis. Epidemiological evidence suggests patients with rheumatoid arthritis are more likely to have periodontitis, which cannot be completely explained by shared risk factors. This has led to the hypothesis that the two diseases are immunologically linked, and that periodontitis may precede, and cause, rheumatoid arthritis. A further objective of this research was to investigate whether the autoimmunity characteristic of rheumatoid arthritis emerges in periodontitis. Results: B cell infiltrate in the gingiva of periodontitis patients was confirmed. Periodontitis patients were found to have elevated serum titers of anti-citrullinated peptide antibodies which were generally below the diagnostic threshold for rheumatoid arthritis, and were reduced following non-surgical periodontal treatment. In a murine model of periodontitis, subtle changes to B cell phenotype were observed in tissues regional to the oral cavity in mice with periodontitis, at an early stage of disease. Such changes included increased B cell expression of receptor activator of NfκB ligand in the gingiva, and increased proportions of GC B cells in the draining lymph nodes. Some of these trends were enhanced in mice with periodontitis exacerbated by interleukin-33 treatment. B cell-deficient mice were protected from the alveolar bone loss normally induced in the model of periodontitis. Conclusion: B cells form a substantial proportion of the inflammatory infiltrate in the gingiva of periodontitis patients. Treatment of periodontitis can reduce titers of anti-citrullinated peptide antibodies in patients, potentially reducing their risk of developing rheumatoid arthritis. Evidence from B cell-deficient mice suggests that B cells contribute to pathological alveolar bone loss. Therefore, B cells may be worthy of targeting therapeutically in periodontitis.