Identifying immune signatures of sepsis to increase diagnostic accuracy in very preterm babies.

Bacterial infections are a major cause of mortality in preterm babies, yet our understanding of early-life disease-associated immune dysregulation remains limited. Here, we combine multi-parameter flow cytometry, single-cell RNA sequencing and plasma analysis to longitudinally profile blood from very preterm babies (<32 weeks gestation) across episodes of invasive bacterial infection (sepsis). We identify a dynamically changing blood immune signature of sepsis, including lymphopenia, reduced dendritic cell frequencies and myeloid cell HLA-DR expression, which characterizes sepsis even when the common clinical marker of inflammation, C-reactive protein, is not elevated. Furthermore, single-cell RNA sequencing identifies upregulation of amphiregulin in leukocyte populations during sepsis, which we validate as a plasma analyte that correlates with clinical signs of disease, even when C-reactive protein is normal. This study provides insights into immune pathways associated with early-life sepsis and identifies immune analytes as potential diagnostic adjuncts to standard tests to guide targeted antibiotic prescribing.

Perinatal inflammation influences but does not arrest rapid immune development in preterm babies.

Infection and infection-related complications are important causes of death and morbidity following preterm birth. Despite this risk, there is limited understanding of the development of the immune system in those born prematurely, and of how this development is influenced by perinatal factors. Here we prospectively and longitudinally follow a cohort of babies born before 32 weeks of gestation. We demonstrate that preterm babies, including those born extremely prematurely (<28 weeks), are capable of rapidly acquiring some adult levels of immune functionality, in which immune maturation occurs independently of the developing heterogeneous microbiome. By contrast, we observe a reduced percentage of CXCL8-producing T cells, but comparable levels of TNF-producing T cells, from babies exposed to in utero or postnatal infection, which precedes an unstable post-natal clinical course. These data show that rapid immune development is possible in preterm babies, but distinct identifiable differences in functionality may predict subsequent infection mediated outcomes.

Unexpected functional consequences of xenogeneic transgene expression in beta-cells of NOD mice.

We describe unexpected alterations in the non-obese diabetic (NOD/Lt) mouse model of type 1 diabetes (T1D) following forced beta-cell expression of non-mammalian genes ligated to an insulin promoter sequence. These include the jellyfish green fluorescent protein (GFP), useful for beta-cell identification, and the bacteriophage P1 Cre recombinase, necessary for beta cell-specific ablation of a gene using a Cre-loxP system. Homozygous expression of GFP, driven by the mouse insulin 1 gene promoter (MIP-GFP) in a single transgenic line of NOD mice, produced T1D in postnatal mice that was not associated with insulitis, but rather beta cell-depleted islets. Hemizygous transgene expression suppressed spontaneous autoimmune T1D in females, and produced a male glucose intolerance syndrome associated with age-dependent declines in plasma insulin content. Among lines of transgenic NOD/Lt mice expressing Cre recombinase driven by the rat insulin 2 promoter (RIP-Cre), high, non-mosaic expression correlated with suppressed T1D development. These findings emphasize the need for careful characterization of genetically manipulated NOD mouse stocks to insure that model characteristics have not been compromised.

The nonhomologous end joining factor Artemis suppresses multi-tissue tumor formation and prevents loss of heterozygosity.

Nonhomologous end joining (NHEJ) is a critical DNA repair pathway, with proposed tumor suppression functions in many tissues. Mutations in the NHEJ factor ARTEMIS cause radiation-sensitive severe combined immunodeficiency in humans and may increase susceptibility to lymphoma in some settings. We now report that deficiency for Artemis (encoded by Dclre1c/Art in mouse) accelerates tumorigenesis in several tissues in a Trp53 heterozygous setting, revealing tumor suppression roles for NHEJ in lymphoid and non-lymphoid cells. We also show that B-lineage lymphomas in these mice undergo loss of Trp53 heterozygosity by allele replacement, but arise by mechanisms distinct from those in Art Trp53 double null mice. These findings demonstrate a general tumor suppression function for NHEJ, and reveal that interplay between NHEJ and Trp53 loss of heterozygosity influences the sequence of multi-hit oncogenesis. We present a model where p53 status at the time of tumor initiation is a key determinant of subsequent oncogenic mechanisms. Because Art deficient mice represent a model for radiation-sensitive severe combined immunodeficiency, our findings suggest that these patients may be at risk for both lymphoid and non-lymphoid cancers.

Combination chemotherapy and IL-15 administration induce permanent tumor regression in a mouse lung tumor model: NK and T cell-mediated effects antagonized by B cells.

Previous studies have demonstrated that IL-15 administration after cyclophosphamide (CY) injection of C57BL/6J mice bearing the i.m. 76-9 rhabdomyosarcoma resulted in a significant prolongation of life. In the present study, we investigated the immune response against the 76-9 experimental lung metastases after CY + IL-15 therapy. Administration of CY + IL-15, but not IL-15 alone, induced prolongation of life and cures in 32% of mice bearing established experimental pulmonary metastases of 76-9 tumor. The CY + IL-15 therapy resulted in increased levels of NK1.1+/LGL-1+ cells, and CD8+/CD44+ T cells in PBL. In vitro cytotoxic assay of PBL indicated the induction of lymphokine-activated killer cell activity, but no evident tumor-specific class I-restricted lytic activity. Survival studies showed that the presence of NK and T lymphocytes is necessary for successful CY + IL-15 therapy. Experiments using knockout mice implied that either alphabeta or gammadelta T cells were required for an antitumor effect induced by CY + IL-15 therapy. However, mice lacking in both alphabeta and gammadelta T cells failed to respond to combination therapy. Cured B6 and alphabeta or gammadelta T cell-deficient mice were immune to rechallenge with 76-9, but not B16LM tumor. B cell-deficient mice showed a significant improvement in the survival rate both after CY and combination CY + IL-15 therapy compared with normal B6 mice. Overall, the data suggest that the interaction of NK cells with tumor-specific alphabeta or gammadelta T lymphocytes is necessary for successful therapy, while B cells appear to suppress the antitumor effects of CY + IL-15 therapy.

CSF-1 regulation of Il6 gene expression by murine macrophages: a pivotal role for GM-CSF.

We test the hypothesis that the monocyte-macrophage colony-stimulating factor (CSF-1 or M-CSF) plays a major role in the inflammatory responses of Mphi by acting as a priming agent that heightens their responsiveness to secondary stimulation by other mediators. We previously reported that CSF-1 induced peritoneal Mphi (PMphi) to transcribe several genes including interleukin-6 (Il6) and granulocyte-macrophage colony-stimulating factor (Csfgm). It was reported that the Il6 and Csfgm genes were individually regulated by different pathways but it was not clear to what extent the two genes interacted during Mphi-mediated inflammatory responses. We now show that CSF-1 induces the release of bioactive GM-CSF from mouse resident PMphi. GM-CSF induces Il6 gene expression and synergizes with CSF-1 to induce the release of large amounts of IL-6. PMphi from C57BL/6J-Csfgm(null) mice were shown to release minimal IL-6 in response to CSF-1 and to express a much reduced response to the highly stimulatory combination of CSF-1 and lipopolysaccharide (LPS). Exogenous recombinant GM-CSF restored the IL-6 response of GM-CSF null PMphi to a great extent but not completely. As controls, three other recombinant proteins were tested but of these only tumor necrosis factor alpha (TNF-alpha) was shown to synergize with both CSF-1 and GM-CSF. Using PMphi from mice deficient in the expression of the Il6 gene, it was shown that they released two- to threefold more GM-CSF in response to CSF-1 than their control counterparts. However, an exogenous supply of recombinant IL-6 had no effect on GM-CSF release. The data indicate that the pathways regulating Il6 gene expression are under the control of a complex network of cytokine interactions involving at least CSF-1, GM-CSF, and TNF-alpha, with the added possibility that IL-6 may exert modulatory activity within this network.

CSF-1 (M-CSF) differentially sensitizes mononuclear phagocyte subpopulations to endotoxin in vivo: a potential pathway that regulates the severity of gram-negative infections.

CSF-1 is known to prime mononuclear phagocytes (MNP) for inflammatory stimuli in vitro. We hypothesized that CSF-1 in vivo can sensitize the host to the increased production of endotoxic shock mediators such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Indeed, when CSF-1-primed mice were challenged with lipopolysaccharide (LPS), increased levels of serum IL-6 and TNF-alpha were detected. Both intravenous and intraperitoneal injections of CSF-1 resulted in increased sensitivity to LPS challenge, which induced maximum increases in serum IL-6 when administered via the intraperitoneal route. The peak serum IL-6 production in control and CSF-1-primed mice occurred 2-3 h after LPS injection, whereas that of TNF-alpha occurred by 1-2 h. When peripheral blood leukocytes, spleen cells, and resident peritoneal cells (PC) were isolated from CSF-1-primed mice injected with LPS, only the PC were shown to release IL-6 constitutively and none released TNF-alpha. A comparison of mRNA isolated from various cells and tissues after intraperitoneal CSF-1 priming indicated that only PC expressed IL-6 mRNA, whereas PC, liver, and spleen expressed TNF-alpha mRNA. All tissues showed increased levels of IL-6 and TNF-alpha mRNA in response to LPS challenge. Only liver and kidney showed an enhanced level of IL-6 expression in CSF-1-primed mice challenged with LPS, whereas liver, lung, and kidney showed enhanced TNF-alpha expression. These data indicate that CSF-1 primes tissue MNP but not circulating MNP to transcribe mRNA and release IL-6 and TNF-alpha. Overall, the data suggest that CSF-1 plays an important role in regulating the sensitivity of the host to the pathophysiological effects of endotoxin.

Essential hypertension in African Caribbeans associates with a variant of the beta2-adrenoceptor.

Populations of West African ancestry dwelling in Western communities exhibit greater prevalence of human essential hypertension and higher rates of end-organ damage. The sympathetic nervous system influences cardiac output, vascular tone, renal sodium reabsorption, and renin release and could be implicated in enhanced vascular responsiveness observed in African hypertensives. Such an effect could arise from genetic variants that alter agonist response of alpha-adrenoceptors, leading to enhanced vasoconstriction, or attenuate beta2-adrenoceptor-mediated vasodilatation. Indeed, there is evidence of a blunted vasodilator response to the beta-agonist isoprenaline in African Americans. A variant of the beta2-adrenoceptor gene that encodes glycine rather than arginine at position 16 (Arg16-->Gly) has been shown to confer exaggerated agonist-mediated receptor downregulation, which might attenuate vasodilator response. One hundred thirty-six unrelated hypertensives and 81 unrelated normotensives of African Caribbean origin were identified from primary care on the island of St Vincent. Genomic DNA from these subjects was analyzed for the presence of the Gly16 and Arg16 alleles by using an allele-specific polymerase chain reaction method. We report strong support for association of the prodownregulatory glycine 16 variant of the beta2-adrenoceptor gene with hypertension in African Caribbeans from St Vincent and the Grenadines (chi2=18.9, P=.000014, 1 df). This observation, coupled with reports of attenuated vasodilator responses to beta-agonists among people of West African ancestry, may provide a mechanism for enhanced vascular reactivity and identify a candidate gene for hypertension in this ethnic group.

Priming of mouse macrophages with the macrophage colony-stimulating factor (CSF-1) induces a variety of pathways that regulate expression of the interleukin 6 (Il6) and granulocyte-macrophage colony-stimulating factor (Csfgm) genes.

Recent data have indicated that resident mouse peritoneal macrophages (PMo) transcribed the interleukin 6 (Il6) and granulocyte-macrophage colony-stimulating factor (Csfgm) genes in response to stimulation with the monocyte-macrophage colony-stimulating factor (CSF-1) but only Il6 mRNA was translated into secreted protein. In this paper, we extend these observations. It is shown that resident PMo incubated with protein kinase (PK)C inhibitors, staurosporine (SP) and its derivative GF109203-X, showed a several fold increase in the levels of Il6 mRNA in control and CSF-1-primed PMo and a parallel release of large amounts of protein. In contrast, SP was shown to have no effect on the release of GM-CSF from control or CSF-1-primed PMo, although it increased by approximately twofold the amount of Csfgm mRNA in CSF-1-primed Mo. When SP was added 4 h after CSF-1 priming to block CSF-1-induced protein kinase pathways, an increased amount of IL-6 release was again seen but without any increase in Il6 mRNA levels. Under these conditions, Csfgm gene expression was relatively unaffected. Activation of PKC by phorbol myristate acetate (PMA) also resulted in increased Il6 gene expression by control and CSF-1-primed PMo. PMA had no apparent effect on Csfgm transcription but appeared to influence translation at a low level, as measured by the release of small amounts of GM-CSF protein. The addition of lipopolysaccharide (LPS) to CSF-1-primed PMo resulted in a synergistic increase in the expression of both genes at the levels of transcription and protein release. The addition of SP to CSF-1-primed Mo before LPS, however, further enhanced IL-6 release but not GM-CSF release from the cells. The data indicate that CSF-1-priming drives a number of pathways involved in the regulation of expression of both genes and renders PMo highly susceptible to appropriate secondary stimulatory agents that transform the PMo into secretory inflammatory cells.

CSF-1-induced and constitutive Il6 gene expression in mouse macrophages: evidence for PKC-dependent and -independent pathways.

It has been recently shown that CSF-1 enhanced the constitutive expression of the Il6 gene in resident mouse peritoneal macrophages (PM phi) but little is known about the pathways involved. In this report, we show that both constitutive and CSF-1-induced IL-6 release were enhanced and prolonged in the presence of the PKC inhibitors, staurosporine (SP) and its derivative, GF-109203X. Enhancement of constitutive IL-6 release required higher concentrations of inhibitors, while enhanced CSF-1-induced release was diminished when inhibitor concentrations exceeded defined limits. SP was also shown to activate constitutive IL-6 release by blood monocytes and elicited PM phi but had no effect on their responsiveness to CSF-1. Activation of PKC by exposure of resident PM phi to phorbol myristate acetate (PMA) also resulted in enhanced IL-6 release and PMA was shown to synergize with CSF-1. These data indicate that CSF-1 does not induce Il6 gene expression by amplifying the constitutive pathway in all mononuclear phagocyte subpopulations. It exerts its effects independently of PKC, which may activate Il6 gene expression in its own right by an alternative pathway. While CSF-1 and PKC are involved in separate pathways, the synergistic IL-6 response seen when PMA and CSF-1 interact suggests convergence of the two pathways. It is also apparent that multiple PKs, excluding PKC, may be involved in repressing constitutive and CSF-1-induced Il6 gene expression.

The atrial natriuretic peptide gene and essential hypertension in African-Caribbeans from St Vincent and the Grenadines.

Atrial natriuretic peptide (ANP) which alters sodium balance, blood volume and vascular tone represents an important candidate for investigating the genetic basis of essential hypertension (EH). Accordingly, we have studied Bgl1 and Xho1 restriction fragment length polymorphisms (RFLPs) of the ANP gene in 147 hypertensive, 141 normotensive and 67 population-based control subjects from a homogenous population of West African origin from St Vincent and the Grenadines. We found no association of either Bgl1 and Xho1 RFLPs with EH. This study suggests that the ANP locus may not exert a major gene effect on EH amongst the black people of St Vincent and the Grenadines.

Angiotensinogen in human essential hypertension.

The candidacy of angiotensinogen for a role in the genetic basis of hypertension is supported by the observation that plasma angiotensinogen levels track with raised blood pressure through families. In addition, transgenic mice with overexpression of a rat angiotensinogen gene develop hypertension, and knockout mice with a disrupted gene and absent angiotensinogen production develop low blood pressure. There are now two studies in populations of white European origin and one in African Caribbeans providing support for a role of the angiotensinogen gene locus in human essential hypertension.

Differential sensitivity of mouse mononuclear phagocytes to CSF-1 and LPS: the potential in vivo relevance of enhanced IL-6 gene expression.

In this report, we compared the responsiveness of subpopulations of mononuclear phagocytes (MNP) to the actions of the monocyte-macrophage colony-stimulating factor (CSF-1) and lipopolysaccharide (LPS), as measured by the expression of the IL-6 (Il6) gene. It was seen that neither monocytes nor elicited peritoneal macrophages (PMphi) responded directly to CSF-1 compared with resident PMphi that were induced to express high levels of Il6 mRNA and release IL-6 protein. Resident PMphi released basal (constitutive) amounts of IL-6, while constitutive release by monocytes and elicited PMphi was barely detectable. Monocytes and elicited PMphi expressed similar levels of sensitivity to LPS, as measured by IL-6 release, and were less reactive than resident PMphi. When CSF-1 and LPS were added simultaneously to resident PMphi, a dose-dependent synergistic release of IL-6 was seen. Elicited PMphi also responded synergistically but required higher levels of CSF-1 and LPS, while monocytes failed to respond synergistically under any conditions. A similar synergistic effect was also seen in vivo when mice were injected with CSF-1 and LPS. Under these conditions, only resident peritoneal cells were shown to release IL-6 ex vivo while blood leukocytes and spleen cells released minimal amounts. These findings indicate that the stage of differentiation/maturation of MNP may be important for the ability of CSF-1 to render the cells sensitive to secondary stimulation, such as by LPS, and determines to what extent MNP subpopulations contribute to inflammatory responses in vivo.

Linkage of the angiotensinogen gene locus to human essential hypertension in African Caribbeans.

The renin-angiotensin system regulates blood pressure and sodium balance. The angiotensinogen gene which encodes the key substrate within this system has been linked to essential hypertension in White Europeans. It has been suggested that people of West African ancestry may have a different genetic basis for hypertension. In this study we have tested whether there is linkage of the angiotensinogen gene to essential hypertension in African Caribbeans from St. Vincent and the Grenadines. DNA from 63 affected sibling pairs with hypertension was tested for linkage by analyzing whether there was excess allele sharing among siblings genotyped using an angiotensinogen dinucleotide repeat sequence. There was significant support for linkage (T = 3.07, P = 0.001) and association of this locus to hypertension (chi 2 = 50.2, 12 degrees of freedom, P << 0.001). A DNA polymorphism which alters methionine to threonine at position 235 (M235T) within the angiotensinogen peptide has been associated previously with hypertension. However, we found no association of this variant with hypertension in this study. These findings provide support for linkage and association of the angiotensinogen locus to hypertension in African Caribbeans and suggest some similarities in the genetic basis of essential hypertension in populations of different ethnicity.

The potential role of the macrophage colony-stimulating factor, CSF-1, in inflammatory responses: characterization of macrophage cytokine gene expression.

In this report we report that recombinant human monocyte-macrophage colony-stimulating factor-1 (CSF-1) induces resident murine peritoneal cells (PCs) to transcribe several inflammatory cytokine genes, including interleukin (IL)-1 alpha, IL-1 beta, IL-6, and granulocyte-macrophage CSF in a dose-dependent and time-related manner. Peak mRNA levels were seen between 4 and 6 h. CSF-1 did not modulate the expression of tumor necrosis factor-alpha mRNA. The serum content of the culture medium appeared to regulate both the extent of CSF-1-induced gene transcription and the adherence properties of the cells. Decreasing the serum concentration significantly reduced CSF-1-induced transcription and was associated with the rapid spreading of the majority of the adherent cells. This reduced sensitivity to CSF-1 was paralleled by a markedly lower levels of c-fms mRNA encoding the CSF-1 receptor. Induced gene transcription was followed by the release of large quantities of IL-6 only. IL-1 activity remained associated with the cells. Neither supernatant nor cell lysate granulocyte-macrophage CSF activity was inducible above the low levels associated with control cultures. Evidence that the mononuclear phagocytes, as opposed to B or T cells, were the targets of CSF-1 was obtained in two ways: (1) PCs from B6 scid/scid and NOD scid/scid mice consisting of 78-86% MAC-1+, F4/80+ cells and few B or T cells, as shown by flow cytometry analysis, released 5- to 10-fold more IL-6 in response to CSF-1 stimulation than B6 PCs, which contained approximately 30% double-positive cells, and (2) pretreatment of B6 PCs with antibodies to the CSF-1 receptor blocked the CSF-1-induced secretion of IL-6. These data suggest that CSF-1 primes noninflammatory mononuclear phagocytes for a role in inflammatory responses but does not provide the necessary signals for either secretion or translation of all cytokines equally.

The therapeutic efficacy of murine anti-tumor T cells: freshly isolated T cells are more therapeutic than T cells expanded in vitro.

Adoptive immunotherapy (AIT) involving transfer of tumor-sensitized T lymphocytes in combination with cyclophosphamide (CY)-injection results in the eradication of the C57BL/6J (B6) rhabdomyosarcoma, 76-9 and is associated with the accumulation of a large number of tumor-infiltrating lymphocytes (TIL). Using immune spleen cells (ISC) from B6 and congenic B6. PL. Thy-1a mice, it was shown that most (> or = 97%) of the TIL were donor-derived. This in situ increase in donor-derived T cells was confirmed by using positively-selected Thy- 1.1+ and Thy- 1.2+ TIL for AIT after isolating them from regressing tumors and expanding them in rIL-2. The extent of CD8+ TIL expansion in vivo correlated with the numbers of TIL adoptively transferred and this in turn determined the degree of anti-tumor effects. It was evident, however, that these in vitro-expanded TIL expressing mRNA for TNF alpha and IFN gamma were qualitatively different and therapeutically less efficacious than the T cells associated with ISC or with freshly-isolated TIL. Unlike freshly isolated TIL that expressed specific cytotoxicity towards the 76-9 targets in vitro, IL-2 expanded TIL killed 76-9 cells and unrelated tumor targets to the same extent. A cytotoxic CD8+ T cell line derived from ISC and selected for activity against the 76-9 tumor cells showed no therapeutic efficacy. The data suggest that, in this tumor model, expansion of CD8+ T cells in vitro selects against anti-tumor efficacy.

Qualitative and quantitative intratumoral changes in gene expression following cyclophosphamide injection and the adoptive transfer of T cells: the potential contribution of tumor-associated macrophages.

Adoptive immunotherapy (AIT) of mice bearing the MCA/76-9 rhabdomyosarcoma in combination with cyclophosphamide (CY) injection results in the permanent regression of tumors. This report is concerned with changes in the tumor-associated macrophage (TAM) population and the influence of both CY injection and CY/AIT on their potential functions. Sequential analyses of FcR, MAC-I and Class-II MHC antigen expressed by tumor-associated cells (TAC) showed that CY injection or CY/AIT induced marked increases in the proportions of all 3 parameters as compared with the relatively stable levels in progressing tumors. These changes were time- and treatment-related. The mean MAC-I fluorescence (antigen density per cell) increased nearly 2-fold by 48 hr after CY injection, regardless of subsequent AIT. In contrast, the density of Class-II antigen per cell declined by as much as 75% within 48 hr after CY injection and did not recover by 7 days. This initial decline was also seen after CY/AIT and was followed by a rapid recovery to near-normal values by day 7. Northern analysis of RNA isolated from whole tumor tissue indicated wide fluctuations in expression of the typical macrophage genes encoding the proteins MAC-I, IL-I alpha, IL-I beta, TNF alpha, IA beta and c-fms. However, with the exception of MAC-I and IL-1 alpha/IL-1 beta mRNA, the modifications appeared to be qualitative rather than representing changes in the proportions of TAM. The data suggest that the changes in membrane antigen and gene expression by TAM reflect a complex interaction between TAM and their environment, in particular tumor cells and tumor-infiltrating lymphocytes. In addition, it is evident that CY injection per se is responsible for defined fundamental changes that presumably influence the outcome of AIT.

Polymorphonuclear leukocyte functions in children with cyanotic and acyanotic congenital heart disease.

Eighteen cyanotic congenital heart disease (CCHD) and 17 acyanotic congenital heart disease (ACHD) patients in the age range of 2 months to 10 years along with their age and nutrition matched controls were studied for bactericidal, chemotactic and phagocytic functions. Bactericidal and phagocytic functions were significantly depressed in CCHD (p < 0.001) as well as ACHD group (p < 0.001) compared with controls. Chemotactic function was not significantly affected in either. Arterial oxygen content (as a measure of hypoxia) was calculated for each patient and correlated with each immune parameter by univariate linear regression analysis. In CCHD patients linear correlation of borderline significance (p = 0.07) was found between arterial oxygen content and bactericidal activity, but no correlation could be established with phagocytic and chemotactic functions. No correlation was obtained between hematocrit and any of the immune parameters. In ACHD patients no correlations were obtained between the immune parameters and arterial oxygen content or hematocrit. Iron deficiency anemia, known to affect bactericidal function, did not seem to affect the immune parameters in CCHD and ACHD groups. Altered oxygen content of the blood owing to hypoxia in CCHD patients may be an important etiological factor in the genesis of bacteremia and cerebral abscess. The affection of immune functions in ACHD cannot be adequately explained.