Host gene expression signatures to identify infection type and organ dysfunction in children evaluated for sepsis: a multicentre cohort study.

BACKGROUND: Sepsis is defined as dysregulated host response to infection that leads to life-threatening organ dysfunction. Biomarkers characterising the dysregulated host response in sepsis are lacking. We aimed to develop host gene expression signatures to predict organ dysfunction in children with bacterial or viral infection. METHODS: This cohort study was done in emergency departments and intensive care units of four hospitals in Queensland, Australia, and recruited children aged 1 month to 17 years who, upon admission, underwent a diagnostic test, including blood cultures, for suspected sepsis. Whole-blood RNA sequencing of blood was performed with Illumina NovaSeq (San Diego, CA, USA). Samples with completed phenotyping, monitoring, and RNA extraction by March 31, 2020, were included in the discovery cohort; samples collected or completed thereafter and by Oct 27, 2021, constituted the Rapid Paediatric Infection Diagnosis in Sepsis (RAPIDS) internal validation cohort. An external validation cohort was assembled from RNA sequencing gene expression count data from the observational European Childhood Life-threatening Infectious Disease Study (EUCLIDS), which recruited children with severe infection in nine European countries between 2012 and 2016. Feature selection approaches were applied to derive novel gene signatures for disease class (bacterial vs viral infection) and disease severity (presence vs absence of organ dysfunction 24 h post-sampling). The primary endpoint was the presence of organ dysfunction 24 h after blood sampling in the presence of confirmed bacterial versus viral infection. Gene signature performance is reported as area under the receiver operating characteristic curves (AUCs) and 95% CI. FINDINGS: Between Sept 25, 2017, and Oct 27, 2021, 907 patients were enrolled. Blood samples from 595 patients were included in the discovery cohort, and samples from 312 children were included in the RAPIDS validation cohort. We derived a ten-gene disease class signature that achieved an AUC of 94·1% (95% CI 90·6-97·7) in distinguishing bacterial from viral infections in the RAPIDS validation cohort. A ten-gene disease severity signature achieved an AUC of 82·2% (95% CI 76·3-88·1) in predicting organ dysfunction within 24 h of sampling in the RAPIDS validation cohort. Used in tandem, the disease class and disease severity signatures predicted organ dysfunction within 24 h of sampling with an AUC of 90·5% (95% CI 83·3-97·6) for patients with predicted bacterial infection and 94·7% (87·8-100·0) for patients with predicted viral infection. In the external EUCLIDS validation dataset (n=362), the disease class and disease severity predicted organ dysfunction at time of sampling with an AUC of 70·1% (95% CI 44·1-96·2) for patients with predicted bacterial infection and 69·6% (53·1-86·0) for patients with predicted viral infection. INTERPRETATION: In children evaluated for sepsis, novel host transcriptomic signatures specific for bacterial and viral infection can identify dysregulated host response leading to organ dysfunction. FUNDING: Australian Government Medical Research Future Fund Genomic Health Futures Mission, Children's Hospital Foundation Queensland, Brisbane Diamantina Health Partners, Emergency Medicine Foundation, Gold Coast Hospital Foundation, Far North Queensland Foundation, Townsville Hospital and Health Services SERTA Grant, and Australian Infectious Diseases Research Centre.

Distinct contributions of the innate immune receptors TLR2 and RP105 to formation and architecture of structured lung granulomas in mice infected with Mycobacterium tuberculosis.

Granulomas are key histopathological features of Mycobacterium tuberculosis (Mtb) infection, with complex roles in pathogen control and dissemination. Thus, understanding drivers and regulators of granuloma formation is important for improving tuberculosis diagnosis, treatment, and prevention. Yet, molecular mechanisms underpinning granuloma formation and dynamics remain poorly understood. Here we used low-dose Mtb infection of C57BL/6 mice, which elicits structured lung granulomas composed of central macrophage clusters encased by a lymphocyte mantle, alongside the disorganized lymphocyte and macrophage clusters commonly observed in Mtb-infected mice. Using gene-deficient mice, we observed that Toll-like receptor (TLR) 2 and the TLR-related Radioprotective 105 kDa protein (RP105) contributed to the extent and spatial positioning of pathology in infected lung tissues, consistent with functional cooperation between TLR2 and RP105 in the innate immune recognition of Mtb. In mice infected with the highly virulent Mtb clinical isolate HN878, TLR2, but not RP105, positively regulated the extent of central macrophage regions within structured granulomas. Moreover, RP105, but not TLR2, promoted the formation of structured lung granulomas, suggesting that the functions of RP105 as an innate immune sensor for Mtb reach beyond its roles as TLR2 co-receptor. TLR2 and RP105 contributions to lung pathology are governed by Mtb biology, as neither receptor affected the frequency or architecture of structured granulomas in mice infected with the reference strain Mtb H37Rv. Thus, by revealing distinctive as well as cooperative functions of TLR2 and RP105 in lung pathology, our data identify TLRs as molecular determinants of TB granuloma formation and architecture, and expand understanding of how interactions between innate immune receptors and Mtb shape TB disease manifestation.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Pre-Diabetes Increases Tuberculosis Disease Severity, While High Body Fat Without Impaired Glucose Tolerance Is Protective.

Type 2 diabetes (T2D) is a well-known risk factor for tuberculosis (TB), but little is known about pre-diabetes and the relative contribution of impaired glucose tolerance vs. obesity towards susceptibility to TB. Here, we developed a preclinical model of pre-diabetes and TB. Mice fed a high fat diet (HFD) for 12 weeks presented with impaired glucose tolerance and hyperinsulinemia compared to mice fed normal chow diet (NCD). Infection with M. tuberculosis (Mtb) H37Rv after the onset of dysglycemia was associated with significantly increased lung pathology, lower concentrations of TNF-α, IFN-γ, IFN-ß and IL-10 and a trend towards higher bacterial burden at 3 weeks post infection. To determine whether the increased susceptibility of pre-diabetic mice to TB is reversible and is associated with dysglycemia or increased body fat mass, we performed a diet reversal experiment. Pre-diabetic mice were fed a NCD for 10 additional weeks (HFD/NCD) at which point glucose tolerance was restored, but body fat mass remained higher compared to control mice that consumed NCD throughout the entire experiment (NCD/NCD). Upon Mtb infection HFD/NCD mice had significantly lower bacterial burden compared to NCD/NCD mice and this was accompanied by restored IFN-γ responses. Our findings demonstrate that pre-diabetes increases susceptibility to TB, but a high body mass index without dysglycemia is protective. This murine model offers the opportunity to further study the underlying immunological, metabolic and endocrine mechanisms of this association.

Functions of the WNT Signaling Network in Shaping Host Responses to Infection.

It is well-established that aberrant WNT expression and signaling is associated with developmental defects, malignant transformation and carcinogenesis. More recently, WNT ligands have emerged as integral components of host responses to infection but their functions in the context of immune responses are incompletely understood. Roles in the modulation of inflammatory cytokine production, host cell intrinsic innate defense mechanisms, as well as the bridging of innate and adaptive immunity have been described. To what degree WNT responses are defined by the nature of the invading pathogen or are specific for subsets of host cells is currently not well-understood. Here we provide an overview of WNT responses during infection with phylogenetically diverse pathogens and highlight functions of WNT ligands in the host defense against infection. Detailed understanding of how the WNT network orchestrates immune cell functions will not only improve our understanding of the fundamental principles underlying complex immune response, but also help identify therapeutic opportunities or potential risks associated with the pharmacological targeting of the WNT network, as currently pursued for novel therapeutics in cancer and bone disorders.

Temporal Regulation of Natural Killer T Cell Interferon Gamma Responses by ß-Catenin-Dependent and -Independent Wnt Signaling.

Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator ß-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. ß-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and ß-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of ß-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of ß-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/ß-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of ß-catenin activity. Our analyses in ICG001-treated mice confirmed a role for ß-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and ß-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of ß-catenin to NKT cell functions.

Roles of WNT, NOTCH, and Hedgehog signaling in the differentiation and function of innate and innate-like lymphocytes.

Innate lymphoid cells (ILCs) and innate-like lymphocytes have important roles in immune responses in the context of infection, cancer, and autoimmunity. The factors involved in driving the differentiation and function of these cell types remain to be clearly defined. There are several cellular signaling pathways involved in embryogenesis, which continue to function in adult tissue. In particular, the WNT, NOTCH, and Hedgehog signaling pathways are emerging as regulators of hematopoietic cell development and differentiation. This review discusses the currently known roles of WNT, NOTCH, and Hedgehog signaling in the differentiation and function of ILCs and innate-like lymphocytes.

TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection.

Neutralization or deletion of tumor necrosis factor (TNF) causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1) expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO) synthase (NOS2) was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg) was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

Fatal Leishmaniasis in the Absence of TNF Despite a Strong Th1 Response.

Induction of inducible nitric oxide synthase in mononuclear phagocytes by IFN-γ and innate tumor necrosis factor (TNF) provide the basis for an effective immune response to the intracellular parasite Leishmania (L.) major. In previous experiments, we observed a fatal visceral form of leishmaniasis in L. major-infected C57BL/6 TNF(-/-) mice. To further delineate the protective function of TNF and its receptor requirements, we comparatively assessed L. major-infected C57BL/6 mice that were either deficient for membrane and soluble TNF (Tnf (-) (/) (-)), for soluble TNF alone (memTnf(Δ/Δ) ), or the TNF receptors type 1 (Tnfr1 (-) (/) (-)) or type 2 (Tnfr2 (-) (/) (-)). We detected locally and systemically increased levels of the cytokine IFN-γ in the absence of the TNF-TNFR1-signaling pathway. An analysis of transcription factors and cytokines revealed that activated Tnf (-) (/) (-) CD4(+) T cells displayed a highly active Th1 phenotype with a strong usage of the T cell receptor Vß5.1/2. From these data we conclude that the fatal outcome of L. major infection in Tnf (-) (/) (-) mice does not result from a skewed or deficient Th1 differentiation.

The absence of CCR7 results in dysregulated monocyte migration and immunosuppression facilitating chronic cutaneous leishmaniasis.

The protozoan parasite Leishmania major causes cutaneous lesions to develop at the site of infection, which are resolved with a strong Th1 immune response in resistant hosts, such as C57BL/6 mice. In contrast, the lesions ulcerate in susceptible hosts which display a Th2 response, such as BALB/c mice. The migration of cells in the immune response to L. major is regulated by chemokines and their receptors. The chemokine receptor CCR7 is expressed on activated DCs and naïve T cells, allowing them to migrate to the correct micro-anatomical positions within secondary lymphoid organs. While there have been many studies on the function of CCR7 during homeostasis or using model antigens, there are very few studies on the role of CCR7 during infection. In this study, we show that B6.CCR7(-/-) mice were unable to resolve the lesion and developed a chronic disease. The composition of the local infiltrate at the lesion was significantly skewed toward neutrophils while the proportion of CCR2(+) monocytes was reduced. Furthermore, a greater percentage of CCR2(+) monocytes expressed CCR7 in the footpad than in the lymph node or spleen of B6.WT mice. We also found an increased percentage of regulatory T cells in the draining lymph node of B6.CCR7(-/-) mice throughout infection. Additionally, the cytokine milieu of the lymph node showed a Th2 bias, rather than the resistant Th1 phenotype. This data shows that CCR7 is required for a protective immune response to intracellular L. major infection.

Different regulatory mechanisms in protozoan parasitic infections.

The immune response to the protozoan pathogens, Leishmania spp., Trypanosoma spp. and Plasmodium spp., has been studied extensively with particular focus on regulation of the immune response by immunological mechanisms. More specifically, in diseases caused by parasites, immunosuppression frequently prevents immunopathology that can injure the host. However, this allows a small number of parasites to evade the immune response and remain in the host after a clinical cure. The consequences can be chronic infections, which establish a zoonotic or anthroponotic reservoir. This review will highlight some of the identified regulatory mechanisms of the immune system that govern immune responses to parasitic diseases, in particular leishmaniasis, trypanosomiasis and malaria, and discuss implications for the development of efficient vaccines against these diseases.