Modeling the Immune Response for Pathogenic and Nonpathogenic Orthohantavirus Infections in Human Lung Microvasculature Endothelial Cells.

Hantaviruses, genus Orthohantavirus, family Hantaviridae, order Bunyavirales, are negative-sense, single-stranded, tri-segmented RNA viruses that persistently infect rodents, shrews, and moles. Of these, only certain virus species harbored by rodents are pathogenic to humans. Infection begins with inhalation of virus particles into the lung and trafficking to the lung microvascular endothelial cells (LMVEC). The reason why certain rodent-borne hantavirus species are pathogenic has long been hypothesized to be related to their ability to downregulate and dysregulate the immune response as well as increase vascular permeability of infected endothelial cells. We set out to study the temporal dynamics of host immune response modulation in primary human LMVECs following infection by Prospect Hill (nonpathogenic), Andes (pathogenic), and Hantaan (pathogenic) viruses. We measured the level of RNA transcripts for genes representing antiviral, proinflammatory, anti-inflammatory, and metabolic pathways from 12 to 72 h with time points every 12 h. Gene expression analysis in conjunction with mathematical modeling revealed a similar profile for all three viruses in terms of upregulated genes that partake in interferon signaling (TLR3, IRF7, IFNB1), host immune cell recruitment (CXCL10, CXCL11, and CCL5), and host immune response modulation (IDO1). We examined secreted protein levels of IFN-ß, CXCL10, CXCL11, CCL5, and IDO in two male and two female primary HLMVEC donors at 48 and 60 h post infection. All three viruses induced similar levels of CCL5, CXCL10, and CXCL11 within a particular donor, and the levels were similar in three of the four donors. All three viruses induced different protein secretion levels for both IFN-ß and IDO and secretion levels differed between donors. In conclusion, we show that there was no difference in the transcriptional profiles of key genes in primary HLMVECs following infection by pathogenic and nonpathogenic hantaviruses, with protein secretion levels being more donor-specific than virus-specific.

Male microbiota-associated metabolite restores macrophage efferocytosis in female lupus-prone mice via activation of PPARγ/LXR signaling pathways.

Systemic lupus erythematosus development is influenced by both sex and the gut microbiota. Metabolite production is a major mechanism by which the gut microbiota influences the immune system, and we have previously found differences in the fecal metabolomic profiles of lupus-prone female and lupus-resistant male BWF1 mice. Here we determine how sex and microbiota metabolite production may interact to affect lupus. Transcriptomic analysis of female and male splenocytes showed genes that promote phagocytosis were upregulated in BWF1 male mice. Because patients with systemic lupus erythematosus exhibit defects in macrophage-mediated phagocytosis of apoptotic cells (efferocytosis), we compared splenic macrophage efferocytosis in vitro between female and male BWF1 mice. Macrophage efferocytosis was deficient in female compared to male BWF1 mice but could be restored by feeding male microbiota. Further transcriptomic analysis of the genes upregulated in male BWF1 mice revealed enrichment of genes stimulated by PPARγ and LXR signaling. Our previous fecal metabolomics analyses identified metabolites in male BWF1 mice that can activate PPARγ and LXR signaling and identified one in particular, phytanic acid, that is a very potent agonist. We show here that treatment of female BWF1 splenic macrophages with phytanic acid restores efferocytic activity via activation of the PPARγ and LXR signaling pathways. Furthermore, we found phytanic acid may restore female BWF1 macrophage efferocytosis through upregulation of the proefferocytic gene CD36. Taken together, our data indicate that metabolites produced by BWF1 male microbiota can enhance macrophage efferocytosis and, through this mechanism, could potentially influence lupus progression.

Feeding lactobacilli impacts lupus progression in (NZBxNZW)F1 lupus-prone mice by enhancing immunoregulation.

Although the relationship between autoimmunity and microorganisms is complex, there is evidence that microorganisms can prevent the development of various autoimmune diseases. Lactobacilli are beneficial gut bacteria that play an important role in immune system development. The goals of this study were to assess the ability of three different strains of lactobacilli (L. casei B255, L. reuteri DSM 17509 and L. plantarum LP299v) to control lupus development/progression in (NZBxNZW)F1 (BWF1) lupus-prone mice before and after disease onset, and identify the mechanisms mediating protection. BWF1 mice fed with individual L. casei or L. reuteri before disease onset exhibited delayed lupus onset and increased survival, while feeding L. plantarum had little impact. In vitro treatment of BWF1 dendritic cells with individual lactobacilli strains upregulated IL-10 production to various extents, with L. casei being the most effective. The protection mediated by L. casei was associated with upregulation of B7-1 and B7-2 by antigen presenting cells, two costimulatory molecules important for regulatory T cell (Treg) induction. Moreover, feeding L. casei lead to increased percentages of CD4+Foxp3+ Tregs and IL10-producing T cells in the lymphoid organs of treated mice. More importantly, mice fed L. casei after disease onset remained stable for several months, i.e. exhibited delayed anti-nucleic acid production and kidney disease progression, and increased survival. Therefore, feeding lactobacilli appears to delay lupus progression possibly via mechanisms involving Treg induction and IL-10 production. Altogether, these data support the notion that ingestion of lactobacilli, with immunoregulatory properties, may be a viable strategy for controlling disease development and progression in patients with lupus, i.e. extending remission length and reducing flare frequency.

Using Multiple Analytical Platforms to Investigate the Androgen Depletion Effects on Fecal Metabolites in a Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by circulating autoantibodies that deposit in target organs (e.g., kidneys), resulting in chronic inflammation and eventual destruction of the organ. SLE is much more prevalent in females than males in both humans and spontaneous mouse models of lupus, such as NZBxNZW F1 (BWF1) mice. Depleting androgens by castration dramatically increases the susceptibility of BWF1 male to lupus. We compared fecal metabolite profiles of castrated BWF1 (androgen-depleted) male, intact (androgen-replete) male, and female mice. Four analytical platforms were employed to study the profiles of polar metabolites in mouse feces collected from adult BWF1 mice, and a total of 435 metabolites was identified. Of these, the abundance levels of 72 metabolites were significantly different between castrated and intact male groups, and 63 metabolites were different between female and male groups. Pathway analysis indicated that the pathway differences between castrated and intact male mice closely resembled the pathway differences between female and intact male mice, suggesting that low levels of androgens, whether due to depletion (castrated male) or endogenous (female), are associated with multiple fecal metabolomic alterations, which could potentially affect SLE progression. Our findings demonstrate that analyzing fecal metabolites using multiple analytical platforms holds great promise for detecting metabolomic alterations in complex disease model systems.

ß-Catenin stabilization in NOD dendritic cells increases IL-12 production and subsequent induction of IFN-γ-producing T cells.

Dendritic cells (DC) from diabetes-prone NOD mice and patients with type 1 diabetes (T1D) produce excess IL-12 that drives development of ß-cell-destroying IFN-γ-producing T cells. The molecular mechanisms that control IL-12 production in T1D are unclear. In this study, we report that ß-catenin, a multifunctional protein involved in inflammation, is dramatically increased in DC from NOD mice. We further investigated the mechanisms leading to accumulation of ß-catenin in NOD DC and its role in the inflammatory pathogenic responses associated with T1D. Hyperphosphorylation of ß-catenin at a stabilizing residue, serine 552, mediated by activation of Akt, appears to lead to ß-catenin accumulation in NOD DC. Elevated ß-catenin in DC correlated with IL-12 production and induction of IFN-γ-producing CD4 cells. On the one hand, knockdown/inhibition of ß-catenin significantly reduced NOD DC production of IL-12 and their ability to induce IFN-γ-producing CD4 cells. On the other hand, overexpression of ß-catenin in control DC resulted in increased IL-12 production and induction of IFN-γ-production in T cells. Additionally, we found that ß-catenin inhibitors decreased NF-κB activation in NOD DC and IFN-γ production by NOD T cells in vivo. These data strongly suggest that accumulation of ß-catenin in DC from NOD mice drives IL-12 production, and consequently, development of pathogenic IFN-γ-producing T cells. Targeting the defect responsible for ß-catenin accumulation and subsequent overproduction of pro-inflammatory cytokines by NOD DC could be an effective therapeutic strategy for the prevention and/or treatment of T1D.

High Thymic Output of Effector CD4+ Cells May Lead to a Treg : T Effector Imbalance in the Periphery in NOD Mice.

Regulatory T cells (Tregs) play a critical role in controlling autoreactive T cells, and quantitative and/or qualitative deficiencies in Tregs are associated with autoimmune diseases, including type 1 diabetes (T1D), in both humans and mice. Both the incidence of T1D and percentages of peripheral Tregs in NOD mice vary considerably between animal facilities. In our animal facility, the incidence of T1D in NOD mice is high at 90-100% and the percentages of peripheral CD4+Foxp3+ cells in ~9-10-week-old female NOD mice are decreased compared to control (B6) mice shortly before high glucose is first detected (~12 weeks). These data suggest that there is an imbalance between Tregs and potentially pathogenic effector T cells at this age that could have significant impact on disease progression to overt diabetes. The goal of the current study was to investigate mechanisms that play a role in peripheral Treg : T effector cell balance in NOD mice, including differences in persistence/survival, peripheral homeostatic proliferation, and thymic production and output of CD4+ T cells. We found no differences in persistence/survival or homeostatic proliferation of either Tregs or effector T cells between NOD and B6 mice. Furthermore, although the percentages and absolute numbers of CD4+Foxp3+ cells in thymus were not decreased in NOD compared to B6 mice, the percentage of CD4+ recent thymic emigrants (RTE) that were Foxp3+ was significantly lower in 9-week-old NOD mice. Interestingly, the thymic output of CD4+Foxp3+ cells was not lower in NOD mice, whereas the thymic output of CD4+Foxp3- cells was significantly higher in NOD mice at that age compared to B6 mice. These data suggest that the higher thymic output of CD4+Foxp3- T cells contributes, at least in part, to the lower percentages of peripheral CD4+Foxp3+ Tregs in NOD mice and an imbalance between Tregs and T effector cells that may contribute to the development of full-blown diabetes.

Relationship between gut microbiota and development of T cell associated disease.

The interplay between the immune response and the gut microbiota is complex. Although it is well-established that the gut microbiota is essential for the proper development of the immune system, recent evidence indicates that the cells of the immune system also influence the composition of the gut microbiota. This interaction can have important consequences for the development of inflammatory diseases, including autoimmune diseases and allergy, and the specific mechanisms by which the gut commensals drive the development of different types of immune responses are beginning to be understood. Furthermore, sex hormones are now thought to play a novel role in this complex relationship, and collaborate with both the gut microbiota and immune system to influence the development of autoimmune disease. In this review, we will focus on recent studies that have transformed our understanding of the importance of the gut microbiota in inflammatory responses.

Tuning of skin immunity by skin commensal bacteria.

Evaluation of: Naik S, Bouladoux N, Wilhelm C et al. Compartmentalized control of skin immunity by resident commensals. Science 337, 1115-1119 (2012). Most analyses of commensal microbiota have been directed toward the gut microbiota and its role in the development of the intestinal immune system, and in regulating the immune response at sites distant from the gut, including the joints or CNS. However, very little is known about how other niches of commensal microbiota affect local immunity and whether they are influenced by the gut microbiota. The current paper reveals that skin commensals are required for the development of protective immunity against a cutaneous pathogen. This immune response driven by skin commensals occurs independently of the gut microbiota and is mediated by MyD88 and IL-1 signaling that promotes protective effector T-cell responses.

Gut microbiota, immunity, and disease: a complex relationship.

Our immune system has evolved to recognize and eradicate pathogenic microbes. However, we have a symbiotic relationship with multiple species of bacteria that occupy the gut and comprise the natural commensal flora or microbiota. The microbiota is critically important for the breakdown of nutrients, and also assists in preventing colonization by potentially pathogenic bacteria. In addition, the gut commensal bacteria appear to be critical for the development of an optimally functioning immune system. Various studies have shown that individual species of the microbiota can induce very different types of immune cells (e.g., Th17 cells, Foxp3(+) regulatory T cells) and responses, suggesting that the composition of the microbiota can have an important influence on the immune response. Although the microbiota resides in the gut, it appears to have a significant impact on the systemic immune response. Indeed, specific gut commensal bacteria have been shown to affect disease development in organs other than the gut, and depending on the species, have been found to have a wide range of effects on diseases from induction and exacerbation to inhibition and protection. In this review, we will focus on the role that the gut microbiota plays in the development and progression of inflammatory/autoimmune disease, and we will also touch upon its role in allergy and cancer.

NOD dendritic cells stimulated with Lactobacilli preferentially produce IL-10 versus IL-12 and decrease diabetes incidence.

Dendritic cells (DCs) from NOD mice produced high levels of IL-12 that induce IFNγ-producing T cells involved in diabetes development. We propose to utilize the microorganism ability to induce tolerogenic DCs to abrogate the proinflammatory process and prevent diabetes development. NOD DCs were stimulated with Lactobacilli (nonpathogenic bacteria targeting TLR2) or lipoteichoic acid (LTA) from Staphylococcus aureus (TLR2 agonist). LTA-treated DCs produced much more IL-12 than IL-10 and accelerated diabetes development when transferred into NOD mice. In contrast, stimulation of NOD DCs with L. casei favored the production of IL-10 over IL-12, and their transfer decreased disease incidence which anti-IL-10R antibodies restored. These data indicated that L. casei can induce NOD DCs to develop a more tolerogenic phenotype via production of the anti-inflammatory cytokine, IL-10. Evaluation of the relative production of IL-10 and IL-12 by DCs may be a very useful means of identifying agents that have therapeutic potential.

Decreased frequencies of CD4+CD25+Foxp3+ cells and the potent CD103+ subset in peripheral lymph nodes correlate with autoimmune disease predisposition in some strains of mice.

The CD4(+)CD25(+)Foxp3(+) cells are essential for regulation of the immune response, and the integrin, CD103 (α(E)ß(7)), identifies a potent subset of these cells. Defects in CD4(+)CD25(+)Foxp3(+) cells are thought to contribute to susceptibility to autoimmune disease in predisposed individuals. Studies evaluating the quality and quantity of CD4(+)CD25(+)Foxp3(+) regulatory cell populations in the context of autoimmune disease susceptibility have been inconclusive, and few if any, have analyzed the CD103 subset. In this study, we analyzed regulatory T cells (Tregs) from different strains of mice with varying degrees of susceptibility to autoimmune disease. We found no differences in the ability of CD4(+)CD25(+) or the CD103(+) subset of Tregs from young female (NZB × NZW)F1 (BWF1), SJL, C57BL/6, or BALB/c mice to suppress CD4(+)CD25(- ) responders in vitro. Analysis of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cell frequencies in lymphoid organs revealed that BWF1 mice had dramatically lower percentages of both populations in the lymph node (LN) than the other strains, and lower percentages in the spleen in all but the C57BL/6 strain. We next determined whether these findings extended to another autoimmune-prone strain. Similar to BWF1 mice, percentages of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cells were significantly lower in predisease NOD mice. The low frequencies of CD4(+)Foxp3(+) and CD4(+)CD25(+)CD103(+) cells in BWF1 and NOD mice were not due to deficiencies in either thymic production or homeostatic proliferation. These data indicate that decreased percentages of CD4(+)Foxp3(+) cells and particularly, CD4(+)CD25(+)CD103(+) cells in LN correlate with the predisposition to spontaneous development of autoimmune disease.

HE3286, an orally bioavailable synthetic analogue of an active DHEA metabolite suppresses spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse.

5-Androstene-3ß,7ß,17ß-triol (AET) is a naturally occurring anti-inflammatory adrenal steroid that limits acute and chronic inflammation. HE3286 (17α-ethynyl-5-androstene-3ß,7ß,17ß-triol) is a synthetic derivative of AET with improved pharmaceutical properties and efficacy in some animal models of autoimmunity. Here, daily oral doses of HE3286 led to a suppression of spontaneous autoimmune diabetes in the non-obese diabetic mouse model of type 1 diabetes mellitus when administered either shortly before or after the first incidence of disease onset. Efficacy was associated with reduced insulitis and a suppression of the pathogenic T helper cell type 1 and type 17 phenotypes in peripheral lymphoid organs. These results demonstrate that daily oral treatment with HE3286 administrated relatively late in the destructive autoimmune process led to a suppression of type 1 diabetes mellitus onset and of the pathological inflammatory status, supporting its clinical evaluation in type 1 diabetes mellitus subjects.

APC activation restores functional CD4(+)CD25(+) regulatory T cells in NOD mice that can prevent diabetes development.

BACKGROUND: Defects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4(+)CD25(+) regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4(+)CD25(+) regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease. METHODOLOGY/PRINCIPAL FINDINGS: To test these hypotheses, we used the well-documented ability of complete Freund's adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4(+)CD25(+) regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4(+)CD25(+) cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4(+)CD25(+)Foxp3(+) cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4(+)CD25(+) cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4(+)CD25(+) cells in vitro compared to APC from untreated NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC.

A novel multimeric form of FasL modulates the ability of diabetogenic T cells to mediate type 1 diabetes in an adoptive transfer model.

Activation induced cell death (AICD) via Fas/FasL is the primary homeostatic molecular mechanism employed by the immune system to control activated T-cell responses and promote tolerance to self-antigens. We herein investigated the ability of a novel multimeric form of FasL chimeric with streptavidin (SA-FasL) having potent apoptotic activity to induce apoptosis in diabetogenic T cells and modulate insulin-dependent type 1 diabetes (IDDM) in an adoptive transfer model. Diabetogenic splenocytes from NOD/Lt females were co-cultured in vitro with SA-FasL, SA control protein, or alone without protein, and adoptively transferred into NOD/Lt-Rag1(null) recipients for diabetes development. All animals receiving control (Alone: n=16 or SA: n=17) cells developed diabetes on average by 6 weeks, whereas animals receiving SA-FasL-treated (n=25) cells exhibited significantly delayed progression (p<.001) and decreased incidence (70%). This effect was associated with an increase in CD4(+)CD25(+) T cells and correlated with FoxP3 expression in pancreatic lymph nodes. Extracorporeal treatment of peripheral blood lymphocytes using SA-FasL during disease onset represents a novel approach that may alter the ability of pathogenic T cells to mediate diabetes and have therapeutic utility in clinical management of IDDM.

Reversing tumor immune suppression with intratumoral IL-12: activation of tumor-associated T effector/memory cells, induction of T suppressor apoptosis, and infiltration of CD8+ T effectors.

A single intratumoral injection of IL-12 and GM-CSF-loaded slow-release microspheres induces T cell-dependent eradication of established primary and metastatic tumors in a murine lung tumor model. To determine how the delivery of cytokines directly to the microenvironment of a tumor nodule induces local and systemic antitumor T cell activity, we characterized therapy-induced phenotypic and functional changes in tumor-infiltrating T cell populations. Analysis of pretherapy tumors demonstrated that advanced primary tumors were infiltrated by CD4+ and CD8+ T cells with an effector/memory phenotype and CD4+CD25+Foxp3+ T suppressor cells. Tumor-associated effector memory CD8+ T cells displayed impaired cytotoxic function, whereas CD4+CD25+Foxp3+ cells effectively inhibited T cell proliferation demonstrating functional integrity. IL-12/GM-CSF treatment promoted a rapid up-regulation of CD43 and CD69 on CD8+ effector/memory T cells, augmented their ability to produce IFN-gamma, and restored granzyme B expression. Importantly, treatment also induced a concomitant and progressive loss of T suppressors from the tumor. Further analysis established that activation of pre-existing effector memory T cells was short-lived and that both the effector/memory and the suppressor T cells became apoptotic within 4 days of treatment. Apoptotic death of pre-existing effector/memory and suppressor T cells was followed by infiltration of the tumor with activated, nonapoptotic CD8+ effector T lymphocytes on day 7 posttherapy. Both CD8+ T cell activation and T suppressor cell purge were mediated primarily by IL-12 and required IFN-gamma. This study provides important insight into how local IL-12 therapy alters the immunosuppressive tumor milieu to one that is immunologically active, ultimately resulting in tumor regression.

Deficiency in NOD antigen-presenting cell function may be responsible for suboptimal CD4+CD25+ T-cell-mediated regulation and type 1 diabetes development in NOD mice.

Various defects in antigen-presenting cells (APCs) and T-cells, including regulatory cells, have been associated with type 1 diabetes development in NOD mice. CD4(+)CD25(+) regulatory cells play a crucial role in controlling various autoimmune diseases, and a deficiency in their number or function could be involved in disease development. The current study shows that NOD mice had fewer CD4(+)CD25(+) regulatory cells, which expressed normal levels of glucocorticoid-induced tumor necrosis factor receptor and cytotoxic T-lymphocyte-associated antigen-4. We have also found that NOD CD4(+)CD25(+) cells regulate poorly in vitro after stimulation with anti-CD3 and NOD APCs in comparison with B6 CD4(+)CD25(+) cells stimulated with B6 APCs. Surprisingly, stimulation of NOD CD4(+)CD25(+) cells with B6 APCs restored regulation, whereas with the reciprocal combination, NOD APCs failed to activate B6 CD4(+)CD25(+) cells properly. Interestingly, APCs from disease-free (>30 weeks of age), but not diabetic, NOD mice were able to activate CD4(+)CD25(+) regulatory function in vitro and apparently in vivo because only spleens of disease-free NOD mice contained potent CD4(+)CD25(+) regulatory cells that prevented disease development when transferred into young NOD recipients. These data suggest that the failure of NOD APCs to activate CD4(+)CD25(+) regulatory cells may play an important role in controlling type 1 diabetes development in NOD mice.

Conversion of CD4+ CD25- cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus.

The CD4+ CD25+ regulatory T cells play a critical role in controlling autoimmunity, but little is known about their development and maintenance. In this study, we investigated whether CD4+ CD25- cells can convert to CD4+ CD25+ regulatory T cells in vivo under natural conditions. CD4+ CD25- cells from CD45.1+ mice were sorted and transferred into congenic CD45.2+ mice. Converted CD4+ CD25+ cells could be detected in lymphoid organs as early as 1 wk after transfer and by 6 wk after transfer, 5-12% of transferred CD4+ cells expressed CD25. Converted CD4+ CD25+ cells themselves failed to proliferate after stimulation, but could suppress proliferation of responder cells in vitro, and also expressed high levels of Foxp3 mRNA. In addition, CD4+ CD25- cells transferred into thymectomized congenic mice converted to CD4+ CD25+ cells that also suppressed responder cell proliferation in vitro, and expressed high levels of Foxp3 mRNA. Finally, CD4+ CD25- cells transferred into B7-/- mice failed to convert into CD4+ CD25+ cells that exhibit the regulatory phenotype. These data indicate that CD4+ CD25- cells convert into CD4+ CD25+ regulatory T cells spontaneously in vivo and suggest that this conversion process could contribute significantly to the maintenance of the peripheral CD4+ CD25+ regulatory T cell population.