Differential production of type I IFN determines the reciprocal levels of IL-10 and proinflammatory cytokines produced by C57BL/6 and BALB/c macrophages

Pattern recognition receptors detect microbial products and induce cytokines, which shape the immunological response. IL-12, TNF-alpha, and IL-1 beta are proinflammatory cytokines, which are essential for resistance against infection, but when produced at high levels they may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine, which dampens proinflammatory responses, but it can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. In this study, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-alpha, and IL-1 beta, but high levels of IL-10, in response to TLR4 and TLR2 ligands LPS and Pam3CSK4, as well as Burkholderia pseudomallei, a Gram-negative bacterium that activates TLR2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN and ERK1/2-dependent, but IL-27-independent, mechanism. Enhanced type I IFN expression in LPS-stimulated C57BL/6 macrophages was accompanied by increased STAT1 and IFN regulatory factor 3 activation. Furthermore, type I IFN contributed to differential IL-1 beta and IL-12 production in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages via both IL-10-dependent and -independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host.his work was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001126), the U.K. Medical Research Council (FC001126), and the Wellcome Trust (FC001126) since April 1, 2015 and before that by U.K. Medical Research Council Grant MRC U117565642 and also by European Research Council Grant 294682-TB-PATH (Crick 10127). A.H. was additionally funded by a U.K. Medical Research Council Centenary Award. M.S. was funded by Fundação para a Ciência e Tecnologia, Portugal Grant FCT-ANR/BIM-MEC/ 0007/2013. M.S. is an associate Fundação para a Ciência e Tecnologia, Portugal investigator.info:eu-repo/semantics/publishedVersio

High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis.

Background: The only available tuberculosis (TB) vaccine, Bacillus Calmette-Guérin (BCG), has variable efficacy. New vaccines are therefore urgently needed. Why BCG fails is incompletely understood, and the tools used for early assessment of new vaccine candidates do not account for BCG variability. Taking correlates of risk of TB disease observed in human studies and back-translating them into mice to create models of BCG variability should allow novel vaccine candidates to be tested early in animal models that are more representative of the human populations most at risk. Furthermore, this could help to elucidate the immunological mechanisms leading to BCG failure. We have chosen the monocyte to lymphocyte (ML) ratio as a correlate of risk of TB disease and have back-translated this into a mouse model. Methods: Four commercially available, inbred mouse strains were chosen. We investigated their baseline ML ratio by flow cytometry; extent of BCG-mediated protection from M ycobacterium tuberculosis infection by experimental challenge; vaccine-induced interferon gamma (IFNγ) response by ELISPOT assay; and tissue distribution of BCG by plating tissue homogenates. Results: The ML ratio varied significantly between A/J, DBA/2, C57Bl/6 and 129S2 mice. A/J mice showed the highest BCG-mediated protection and lowest ML ratio, while 129S2 mice showed the lowest protection and higher ML ratio. We also found that A/J mice had a lower antigen specific IFNγ response than 129S2 mice. BCG tissue distribution appeared higher in A/J mice, although this was not statistically significant. Conclusions: These results suggest that the ML ratio has an impact on BCG-mediated protection in mice, in alignment with observations from clinical studies. A/J and 129S2 mice may therefore be useful models of BCG vaccine variability for early TB vaccine testing. We speculate that failure of BCG to protect from TB disease is linked to poor tissue distribution in a ML high immune environment

Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates

CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis.

Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients

Examining time-varying dynamics of co-occurring depressed mood and social anxiety

Dimensional frameworks of psychopathology (e.g., HiTOP models) call for multivariate approaches to map co-occurring symptoms clustered within spectra to index what symptoms emerge when and for whom. However, previous work measuring symptom dynamics and affective experiences has largely neglected transdiagnostic measurement of how symptoms co-vary between- and within-individuals over time. Ecological momentary assessment (EMA) offers a method for assessing and differentiating the dynamics of co-occurring symptoms with greater temporal granularity and naturalistic context. The present study used multivariate mixed effects location-scale modeling to characterize the time-varying dynamics of depressed mood and anxiety for women diagnosed with social anxiety disorder, major depression, and related co-occurring diagnoses. Women completed five daily EMA surveys over 30 days (150 EMA surveys/woman, T 5,250 total observations) and two clinical diagnostic and self-report batteries conducted approximately two months apart. There was evidence of lagged effects within-factor (bs = .08-.09), but not cross-factor (bs < .01) during EMA. Symptoms co-varied such that momentary spikes from one’s typical level of anxiety were associated with increases in momentary depressed mood (b = .19) and greater volatility of depressed mood (b = .06). Similarly, spikes from one’s typical levels of depressed mood were associated with increases in momentary anxiety (b = .19). Furthermore, the presence and magnitude of effects demonstrated person-specific heterogeneity. Findings from this work help to characterize how daily experiences of co-occurring mood and anxiety fluctuate and offer insight to aid the development of momentary, person-specific interventions designed to regulate symptom fluctuations

T Cell Immunity to the Alkyl Hydroperoxide Reductase of Burkholderia pseudomallei: A Correlate of Disease Outcome in Acute Melioidosis.

There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of 'humanized' HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection

Identification of an OmpW homologue in Burkholderia pseudomallei, a protective vaccine antigen against melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis, which is associated with a range of clinical manifestations, including sepsis and fatal pneumonia and is endemic in Southeast Asia and Northern Australia. Treatment can be challenging and control of infection involves prolonged antibiotic therapy, yet there are no approved vaccines available to prevent infection. Our aim was to develop and assess the potential of a prophylactic vaccine candidate targeted against melioidosis. The identified candidate is the 22kDa outer membrane protein, OmpW. We previously demonstrated that this protein was immunoprotective in mouse models of Burkholderia cepacia complex (Bcc) infections. We cloned Bp_ompW in Escherichia coli, expressed and purified the protein. Endotoxin free protein administered with SAS adjuvant protected Balb/C mice (75% survival) relative to controls (25% survival) (p<0.05). A potent serological response was observed with IgG2a to IgG1 ratio of 6.0. Furthermore C57BL/6 mice were protected for up to 80 days against a lethal dose of B. pseudomallei and surpassed the efficacy of the live attenuated 2D2 positive control. BpompW is homologous across thirteen sequenced B. pseudomallei strains, indicating that it should be broadly protective against B. pseudomallei. In conclusion, we have demonstrated that BpOmpW is able to induce protective immunity against melioidosis and is likely to be an effective vaccine antigen, possibly in combination with other subunit antigens

The Blood Transcriptome of Experimental Melioidosis Reflects Disease Severity and Shows Considerable Similarity with the Human Disease.

Melioidosis, a severe human disease caused by the bacterium Burkholderia pseudomallei, has a wide spectrum of clinical manifestations ranging from acute septicemia to chronic localized illness or latent infection. Murine models have been widely used to study the pathogenesis of infection and to evaluate novel therapies or vaccines, but how faithfully they recapitulate the biology of human melioidosis at a molecular level is not known. In this study, mice were intranasally infected with either high or low doses of B. pseudomallei to generate either acute, chronic, or latent infection and host blood and tissue transcriptional profiles were generated. Acute infection was accompanied by a homogeneous signature associated with induction of multiple innate immune response pathways, such as IL-10, TREM1, and IFN signaling, largely found in both blood and tissue. The transcriptional profile in blood reflected the heterogeneity of chronic infection and quantitatively reflected the severity of disease. Genes associated with fibrosis and tissue remodeling, including matrix metalloproteases and collagen, were upregulated in chronically infected mice with severe disease. Transcriptional signatures of both acute and chronic melioidosis revealed upregulation of iNOS in tissue, consistent with the expression of IFN-γ, but also Arginase-1, a functional antagonist of the iNOS pathway, and was confirmed by immunohistochemistry. Comparison of these mouse blood datasets by pathway and modular analysis with the blood transcriptional signature of patients with melioidosis showed that many genes were similarly perturbed, including Arginase-1, IL-10, TREM1, and IFN signaling, revealing the common immune response occurring in both mice and humans