Heat shock factor 1 drives regulatory T-cell induction to limit murine intestinal inflammation.

The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of clusters of differentiation (CD)4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and transforming growth factor ß, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using chromatin immunoprecipitation (ChIP), we demonstrate the direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when wild-type Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T-cell component. SIGNIFICANCE STATEMENT: The heat shock response (HSR) is a canonical stress response triggered by a multitude of stressors, including inflammation. Evidence supports the role of the HSR in regulating inflammation, yet there is a paucity of data on its influence in T cells specifically. Gut homeostasis reflects a balance between regulatory clusters of differentiation (CD)4+ T cells and pro-inflammatory T-helper (Th)17 cells. We show that upon activation within T cells, heat shock factor 1 (HSF1) translocates to the nucleus, and stimulates Treg-specific gene expression. HSF1 deficiency hinders Treg development and function and conversely, HSF1 overexpression enhances Treg development and function. While this work, focuses on HSF1 as a novel therapeutic target for intestinal inflammation, the findings have significance for a broad range of inflammatory conditions.

The TNFΔARE Mouse as a Model of Intestinal Fibrosis.

Crohn disease (CD) is a highly morbid chronic inflammatory disease. Although many patients with CD also develop fibrostenosing complications, there are no medical therapies for intestinal fibrosis. This is due, in part, to a lack of high-fidelity biomimetic models to enhance understanding and drug development, which highlights the need for developing in vivo models of inflammatory bowel disease-related intestinal fibrosis. This study investigates whether the TNFΔARE mouse, a model of ileal inflammation, also develops intestinal fibrosis. Several clinically relevant outcomes were studied, including features of structural fibrosis, histologic fibrosis, and gene expression. These include the use of a new luminal casting technique, traditional histologic outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNFΔARE mice as well as in cohorts of numerous ages. At >24 weeks of age, TNFΔARE mice developed structural, histologic, and transcriptional changes of ileal fibrosis. Protein and RNA expression profiles showed changes as early as 6 weeks, coinciding with histologic changes as early as 14 to 15 weeks. Overt structural fibrosis was delayed until at least 16 weeks and was most developed after 24 weeks. This study found that the TNFΔARE mouse is a viable and highly tractable model of ileal fibrosis. This model and the techniques used herein can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

Chemokine Signatures of Pathogen-Specific T Cells II: Memory T Cells in Acute and Chronic Infection.

Pathogen-specific memory T cells (TM) contribute to enhanced immune protection under conditions of reinfection, and their effective recruitment into a recall response relies, in part, on cues imparted by chemokines that coordinate their spatiotemporal positioning. An integrated perspective, however, needs to consider TM as a potentially relevant chemokine source themselves. In this study, we employed a comprehensive transcriptional/translational profiling strategy to delineate the identities, expression patterns, and dynamic regulation of chemokines produced by murine pathogen-specific TM CD8+TM, and to a lesser extent CD4+TM, are a prodigious source for six select chemokines (CCL1/3/4/5, CCL9/10, and XCL1) that collectively constitute a prominent and largely invariant signature across acute and chronic infections. Notably, constitutive CCL5 expression by CD8+TM serves as a unique functional imprint of prior antigenic experience; induced CCL1 production identifies highly polyfunctional CD8+ and CD4+TM subsets; long-term CD8+TM maintenance is associated with a pronounced increase of XCL1 production capacity; chemokines dominate the earliest stages of the CD8+TM recall response because of expeditious synthesis/secretion kinetics (CCL3/4/5) and low activation thresholds (CCL1/3/4/5/XCL1); and TM chemokine profiles modulated by persisting viral Ags exhibit both discrete functional deficits and a notable surplus. Nevertheless, recall responses and partial virus control in chronic infection appear little affected by the absence of major TM chemokines. Although specific contributions of TM-derived chemokines to enhanced immune protection therefore remain to be elucidated in other experimental scenarios, the ready visualization of TM chemokine-expression patterns permits a detailed stratification of TM functionalities that may be correlated with differentiation status, protective capacities, and potential fates.

Chemokine Signatures of Pathogen-Specific T Cells I: Effector T Cells.

The choreography of complex immune responses, including the priming, differentiation, and modulation of specific effector T cell populations generated in the immediate wake of an acute pathogen challenge, is in part controlled by chemokines, a large family of mostly secreted molecules involved in chemotaxis and other patho/physiological processes. T cells are both responsive to various chemokine cues and a relevant source for certain chemokines themselves; yet, the actual range, regulation, and role of effector T cell-derived chemokines remains incompletely understood. In this study, using different in vivo mouse models of viral and bacterial infection as well as protective vaccination, we have defined the entire spectrum of chemokines produced by pathogen-specific CD8+ and CD4+T effector cells and delineated several unique properties pertaining to the temporospatial organization of chemokine expression patterns, synthesis and secretion kinetics, and cooperative regulation. Collectively, our results position the "T cell chemokine response" as a notably prominent, largely invariant, yet distinctive force at the forefront of pathogen-specific effector T cell activities and establish novel practical and conceptual approaches that may serve as a foundation for future investigations into the role of T cell-produced chemokines in infectious and other diseases.

Aging boosts antiviral CD8+T cell memory through improved engagement of diversified recall response determinants.

The determinants of protective CD8+ memory T cell (CD8+TM) immunity remain incompletely defined and may in fact constitute an evolving agency as aging CD8+TM progressively acquire enhanced rather than impaired recall capacities. Here, we show that old as compared to young antiviral CD8+TM more effectively harness disparate molecular processes (cytokine signaling, trafficking, effector functions, and co-stimulation/inhibition) that in concert confer greater secondary reactivity. The relative reliance on these pathways is contingent on the nature of the secondary challenge (greater for chronic than acute viral infections) and over time, aging CD8+TM re-establish a dependence on the same accessory signals required for effective priming of naïve CD8+T cells in the first place. Thus, our findings reveal a temporal regulation of complementary recall response determinants that is consistent with the recently proposed "rebound model" according to which aging CD8+TM properties are gradually aligned with those of naïve CD8+T cells; our identification of a broadly diversified collection of immunomodulatory targets may further provide a foundation for the potential therapeutic "tuning" of CD8+TM immunity.

Aging of Antiviral CD8+ Memory T Cells Fosters Increased Survival, Metabolic Adaptations, and Lymphoid Tissue Homing.

Aging of established antiviral T cell memory can foster a series of progressive adaptations that paradoxically improve rather than compromise protective CD8+ T cell immunity. We now provide evidence that this gradual evolution, the pace of which is contingent on the precise context of the primary response, also impinges on the molecular mechanisms that regulate CD8+ memory T cell (TM) homeostasis. Over time, CD8+ TM generated in the wake of an acute infection with the natural murine pathogen lymphocytic choriomeningitis virus become more resistant to apoptosis and acquire enhanced cytokine responsiveness without adjusting their homeostatic proliferation rates; concurrent metabolic adaptations promote increased CD8+ TM quiescence and fitness but also impart the reacquisition of a partial effector-like metabolic profile; and a gradual redistribution of aging CD8+ TM from blood and nonlymphoid tissues to lymphatic organs results in CD8+ TM accumulations in bone marrow, splenic white pulp, and, particularly, lymph nodes. Altogether, these data demonstrate how temporal alterations of fundamental homeostatic determinants converge to render aged CD8+ TM poised for greater recall responses.

C/EBPß Deletion Promotes Expansion of Poorly Functional Intestinal Regulatory T Cells.

BACKGROUND AND AIMS: Inflammatory Bowel Diseases [IBDs] are chronic intestinal inflammatory conditions in part mediated by CD4+ T cells. Anti-inflammatory Foxp3+ regulatory T cells [Tregs] maintain immune homeostasis and protect against IBD development via multiple mechanisms, including cytokine secretion and cell-cell interaction. CCAAT enhancer binding protein-beta [C/EBPß] is a stress-responsive transcription factor linked with IBD susceptibility. Whole-body C/EBPß deficiency induces CD4+ T cell-predominant hyperproliferation, and we hypothesize that this may be due to impaired Treg function. METHODS: We used the C/EBPß-/- mice in the CD45RBHigh adoptive transfer model, to assess C/EBPß-/- CD4+ T cells for their colitiogenic potential, and C/EBPß-/- CD4+ Foxp3+ Tregs for their ability to inhibit colitis. We assessed Tregs from the C/EBPß-/- mice for expression of Treg functional genes and proteins. RESULTS: Naïve C/EBPß-/- CD4+ T cells are more colitogenic in vivo. The exacerbated colitis does not appear to reflect impaired Treg development, however, as C/EBPß-/- mice displayed more, rather than fewer intestinal CD4+Foxp3+ Tregs in vivo. Instead, this reflects impaired Treg function as seen by the reduced capacity to suppress T cell proliferation in vitro, along with decreased secretion of the anti-inflammatory cytokine IL-10. These findings were corroborated in vivo by additional adoptive co-transfer studies in which wildtype Tregs prevented colitis but C/EBPß-/- Tregs did not. CONCLUSION: C/EBPß deficiency impairs Treg function and potentiates T cell-mediated colitis. A clearer understanding of the function of this transcription factor may provide a novel therapeutic strategy for IBD.

VEN-120, a Recombinant Human Lactoferrin, Promotes a Regulatory T Cell [Treg] Phenotype and Drives Resolution of Inflammation in Distinct Murine Models of Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is characterised by a disruption of immune homeostasis, which is tightly regulated to protect against harmful pathogens yet not react to commensal antigens. Animal studies indicate that regulatory T cells [Treg] modulate the immune response to prevent IBD development. Lactoferrin [LF] is an endogenous anti-inflammatory pleiotropic protein secreted at high concentrations in colostrum and at mucosal sites. However, the effect of LF on specific T lymphocyte populations has not been studied. Here, we identify a novel mechanism by which a recombinant human LF, VEN-120, regulates T cell populations in health and disease. METHODS: Two murine models of intestinal inflammation, the dextran sodium sulphate colitis model and the TNFΔARE/+ model of ileitis, were used to study the anti-inflammatory and T cell modulating ability of VEN-120. Flow cytometry was used to evaluate T cell populations within the lamina propria and mesenteric lymph nodes, and to evaluate the effect of VEN-120 on CD4+ T cells in vitro. RESULTS: VEN-120 reduced inflammation in both models of IBD, accompanied by increased Tregs in the intestinal lamina propria. Treatment of CD4+ T cells in vitro resulted in an upregulation of Treg genes and skewing towards a Treg population. This in vitro T cell skewing translated to an increase of Treg homing to the intestinal lamina propria and associated lymph tissue in healthy mice. CONCLUSIONS: These data provide a novel immunological mechanism by which VEN-120 modulates T cells to restrict inflammatory T cell-driven disease.

Aspergillus fumigatus invasion increases with progressive airway ischemia.

Despite the prevalence of Aspergillus-related disease in immune suppressed lung transplant patients, little is known of the host-pathogen interaction. Because of the mould's angiotropic nature and because of its capacity to thrive in hypoxic conditions, we hypothesized that the degree of Aspergillus invasion would increase with progressive rejection-mediated ischemia of the allograft. To study this relationship, we utilized a novel orthotopic tracheal transplant model of Aspergillus infection, in which it was possible to assess the effects of tissue hypoxia and ischemia on airway infectivity. Laser Doppler flowmetry and FITC-lectin were used to determine blood perfusion, and a fiber optic microsensor was used to measure airway tissue oxygen tension. Fungal burden and depth of invasion were graded using histopathology. We demonstrated a high efficacy (80%) for producing a localized fungal tracheal infection with the majority of infection occurring at the donor-recipient anastomosis; Aspergillus was more invasive in allogeneic compared to syngeneic groups. During the study period, the overall kinetics of both non-infected and infected allografts was similar, demonstrating a progressive loss of perfusion and oxygenation, which reached a nadir by days 10-12 post-transplantation. The extent of Aspergillus invasion directly correlated with the degree of graft hypoxia and ischemia. Compared to the midtrachea, the donor-recipient anastomotic site exhibited lower perfusion and more invasive disease; a finding consistent with clinical experience. For the first time, we identify ischemia as a putative risk factor for Aspergillus invasion. Therapeutic approaches focused on preserving vascular health may play an important role in limiting Aspergillus infections.

High efficiency of antiviral CD4(+) killer T cells.

The destruction of infected cells by cytotxic T lymphocytes (CTL) is integral to the effective control of viral and bacterial diseases, and CTL function at large has long been regarded as a distinctive property of the CD8(+)T cell subset. In contrast, and despite their first description more than three decades ago, the precise contribution of cytotoxic CD4(+)T cells to the resolution of infectious diseases has remained a matter of debate. In particular, the CTL activity of pathogen-specific CD4(+) "helper" T cells constitutes a single trait among a diverse array of other T cell functionalities, and overall appears considerably weaker than the cytolytic capacity of CD8(+) effector T cells. Here, using an in vivo CTL assay, we report that cytotoxic CD4(+)T cells are readily generated against both viral and bacterial pathogens, and that the efficiency of MHC-II-restricted CD4(+)T cell killing adjusted for effector:target cell ratios, precise specificities and functional avidities is comparable in magnitude to that of CD8(+)T cells. In fact, the only difference between specific CD4(+) and CD8(+)T cells pertains to the slightly delayed killing kinetics of the former demonstrating that potent CTL function is a cardinal property of both antiviral CD8(+) and CD4(+)T cells.

Expression and regulation of chemokines in murine and human type 1 diabetes.

More than one-half of the ~50 human chemokines have been associated with or implicated in the pathogenesis of type 1 diabetes, yet their actual expression patterns in the islet environment of type 1 diabetic patients remain, at present, poorly defined. Here, we have integrated a human islet culture system, murine models of virus-induced and spontaneous type 1 diabetes, and the histopathological examination of pancreata from diabetic organ donors with the goal of providing a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family. Chemokine (C-C motif) ligand (CCL) 5 (CCL5), CCL8, CCL22, chemokine (C-X-C motif) ligand (CXCL) 9 (CXCL9), CXCL10, and chemokine (C-X3-C motif) ligand (CX3CL) 1 (CX3CL1) were the major chemokines transcribed (in an inducible nitric oxide synthase-dependent but not nuclear factor-κB-dependent fashion) and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients, whereas CCL5, CCL8, CXCL9, and CX3CL1 proteins were present at lower levels in the islets of both species. Of importance, additional expression of the same chemokines in human acinar tissues emphasizes an underappreciated involvement of the exocrine pancreas in the natural course of type 1 diabetes that will require consideration for additional type 1 diabetes pathogenesis and immune intervention studies.

Multiple layers of CD80/86-dependent costimulatory activity regulate primary, memory, and secondary lymphocytic choriomeningitis virus-specific T cell immunity.

The lymphocytic choriomeningitis virus (LCMV) system constitutes one of the most widely used models for the study of infectious disease and the regulation of virus-specific T cell immunity. However, with respect to the activity of costimulatory and associated regulatory pathways, LCMV-specific T cell responses have long been regarded as relatively independent and thus distinct from the regulation of T cell immunity directed against many other viral pathogens. Here, we have reevaluated the contribution of CD28-CD80/86 costimulation in the LCMV system by use of CD80/86-deficient mice, and our results demonstrate that a disruption of CD28-CD80/86 signaling compromises the magnitude, phenotype, and/or functionality of LCMV-specific CD8(+) and/or CD4(+) T cell populations in all stages of the T cell response. Notably, a profound inhibition of secondary T cell immunity in LCMV-immune CD80/86-deficient mice emerged as a composite of both defective memory T cell development and a specific requirement for CD80 but not CD86 in the recall response, while a related experimental scenario of CD28-dependent yet CD80/86-independent secondary CD8(+) T cell immunity suggests the existence of a CD28 ligand other than CD80/86. Furthermore, we provide evidence that regulatory T cells (T(REG)s), the homeostasis of which is altered in CD80/86(-/-) mice, contribute to restrained LCMV-specific CD8(+) T cell responses in the presence of CD80/86. Our observations can therefore provide a more coherent perspective on CD28-CD80/86 costimulation in antiviral T cell immunity that positions the LCMV system within a shared context of multiple defects that virus-specific T cells acquire in the absence of CD28-CD80/86 costimulation.

Comprehensive assessment of chemokine expression profiles by flow cytometry.

The chemokines are a large family of mainly secreted molecules involved in the regulation of numerous physiological and pathophysiological processes. Despite many years of investigation, the precise cellular sources of most chemokines have remained incompletely defined as a consequence of the limited availability of suitable reagents to visualize the expression of chemokine proteins at the single-cell level. Here, we developed a simple flow cytometry-based assay using commercially available chemokine-specific antibodies for efficient cell-associated detection of 37 of 39 murine chemokines. To demonstrate the utility of this methodology, we used it to reevaluate the nature of homeostatic chemokines in the hematopoietic compartment, to delineate the complete chemokine profiles of NK cells and B cells in response to major polyclonal stimuli, and to assess the chemokine response of DCs to bacterial infection. The versatility of this analytical methodology was further demonstrated by its application to selected human chemokines and should greatly facilitate any future investigation into chemokine biology at large.