Stephen J. Thompson

TRPV1 Deletion Enhances Local Inflammation and Accelerates the Onset of Systemic Inflammatory Response Syndrome

The transient receptor potential vanilloid 1 (TRPV1) is primarily localized to sensory nerve fibers and is associated with the stimulation of pain and inflammation. TRPV1 knockout (TRPV1KO) mice show enhanced LPS-induced sepsis compared with wild type (WT). This implies that TRPV1 may have a key modulatory role in increasing the beneficial and reducing the harmful components in sepsis. We investigated immune and inflammatory mechanisms in a cecal ligation and puncture (CLP) model of sepsis over 24 h. CLP TRPV1KO mice exhibited significant hypothermia, hypotension, and organ dysfunction compared with CLP WT mice. Analysis of the inflammatory responses at the site of initial infection (peritoneal cavity) revealed that CLP TRPV1KO mice exhibited: 1) decreased mononuclear cell integrity associated with apoptosis, 2) decreased macrophage tachykinin NK1-dependent phagocytosis, 3) substantially decreased levels of nitrite (indicative of NO) and reactive oxygen species, 4) increased cytokine levels, and 5) decreased bacteria clearance when compared with CLP WT mice. Therefore, TRPV1 deletion is associated with impaired macrophage-associated defense mechanisms. Thus, TRPV1 acts to protect against the damaging impact of sepsis and may influence the transition from local to a systemic inflammatory state.

Systemic administration of tolerogenic dendritic cells ameliorates murine inflammatory arthritis.

The expression of various cell surface molecules and the production of certain cytokines are important mechanisms by which dendritic cells (DC) are able to bias immune responses. This paper describes the effects of the inflammatory cytokine tumor necrosis factor (TNF)-α on DC phenotype and function. TNF-α treatment resulted in upregulation of MHC class II and CD86 in the absence of increased cell surface CD40 and CD80 or the production of IL-12. Additionally TNF-α treated cells were able to bias T cell responses towards an anti-inflammatory profile. On a note of caution this tolerogenic phenotype of the DC was not stable upon subsequent TLR-4 ligation as a 4 hour pulse of the TNF-α treated DC with lipopolysaccharide (LPS) resulted in the restoration of IL-12 production and an enhancement of their T cell stimulatory capacity which resulted in an increased IFN-γ production. However, TNF-α treated DC, when administered in vivo, were shown to ameliorate disease in collagen induced arthritis, an experimental model of inflammatory joint disease. Mice receiving TNF-α treated DC but not LPS matured DC had a delayed onset, and significantly reduced severity, of arthritis. Disease suppression was associated with reduced levels of collagen specific IgG2a and decreased inflammatory cell infiltration into affected joints. In summary the treatment of DC with TNF-α generates an antigen presenting cell with a phenotype that can reduce the pro-inflammatory response and direct the immune system towards a disease modifying, anti-inflammatory state.

Pro-resolution immunological networks: binding immunoglobulin protein and other resolution-associated molecular patterns

Appropriate regulation and subsequent resolution of acute inflammatory events is critical to the prevention of autoinflammatory diseases. Indeed, the chronic inflammation observed in diseases such as RA is at least partially consequent on the failure of endogenous immunoregulation. Current RA therapies (e.g. anti-TNF-α inhibitors and MTX) inhibit components of the inflammatory disease process without directly promoting the resolution of inflammation. We propose that the next generation of RA therapeutics will complement and augment endogenous immunoregulatory and pro-resolution immunological networks, thus promoting the definitive resolution of inflammation rather than temporary immunological control. Of particular interest with respect to this therapeutic approach is binding immunoglobulin protein [BiP; also known as glucose-regulated protein-78 (GRP78)], a member of the recently defined resolution-associated molecular pattern (RAMP) family of molecules. In this review, we consider the preclinical evidence from experiments in mouse and man that suggests BiP and other members of the RAMP family have the potential to herald a new generation of immunotherapeutics.

Increased percentages of regulatory T cells are associated with inflammatory and neuroendocrine responses to acute psychological stress and poorer health status in older men and women

RationaleThe percentage of regulatory T cells (TRegs)—a subtype of T lymphocyte that suppresses the immune response—appears to be reduced in a number of stress-related diseases. The role of the TReg in stress-disease pathways has not yet been investigated.ObjectivesThe aim of the study was to investigate the association between biological responsivity to acute psychosocial stress and the percentage of TRegs in healthy older adults. The secondary purpose was to measure the associations between TReg percentage and psychological and physical well-being in the participants.MethodsSalivary cortisol and plasma interleukin (IL)-6 samples were obtained from 121 healthy older men and women from the Whitehall II cohort following acute psychophysiological stress testing. Three years later at a follow-up visit, we measured TReg percentages and psychological and physical well-being were recorded using the Short Form 36 Health Survey and the Center for Epidemiologic Studies Depression Scale.ResultsBlunted cortisol responses (pu2009=u20090.004) and elevated IL-6 responses (pu2009=u20090.027) to acute psychophysiological stress were associated with greater TReg percentage independently of age, sex, BMI, smoking status, employment grade, time of testing, and baseline measures of cortisol and IL-6, respectively. Percentage of TRegs was associated cross-sectionally with lower physical (pu2009=u20090.043) and mental health status (pu2009=u20090.008), and higher levels of depressive symptoms (pu2009=u20090.002), independently of covariates.ConclusionsIncreased levels of TRegs may act as a defence against increased inflammation and may be a pre-indication for chronically stressed individuals on the cusp of clinical illness.

Heat-Shock Proteins in Autoimmunity

Heat shock proteins (HSPs), also known as “stress proteins,” are among the highly conserved and immunogenic proteins shared among diverse groups of microbial agents and mammals [1]. Heat and other types of stressful stimuli can increase the cellular expression of HSPs. These proteins have been categorized into different families according to their molecular mass, for example, HSP110, HSP90, HSP70, HSP60, HSP40, HSP20-30, and HSP10 [1–3]. For uniformity, guidelines for the nomenclature of various human HSP families have been proposed [4]. Under physiological conditions, the ubiquitously distributed HSPs maintain the integrity and function of other cellular proteins in stressful conditions. However, HSPs also can become targets of immune response, resulting in immune pathology and clinical manifestations of autoimmunity. HSPs have been implicated in the pathogenesis of a variety of autoimmune diseases [3, 5–7]. Moreover, the involvement of other small HSPs (e.g., H11/HspB8) besides the canonical members of that family in health and disease is increasingly being realized. Importantly, HSPs are also capable of inducing immune responses that are immunoregulatory in nature [7–10], including resolution of the inflammatory responses [11]. Also, the concept of immune network (immunological homunculus) including HSPs as one of its components has been proposed to explain immune homeostasis in health and disease [12]. n nThe main focus of this special issue is on the role of different HSPs in the pathogenesis of autoimmunity through induction/propagation as well as regulation of the disease-related processes; on the impact of physiological and disease-related metabolic processes on the induction of HSPs; on the mechanistic basis of the effector functions driven by HSPs; and on the immunomodulatory role of HSPs. Thirteen excellent papers describing new original results and the most recent developments in the field on these topics are presented in this special issue. These papers cover the role of HSPs in antigen cross-presentation, induction of autoimmunity, and immunotherapy of autoimmunity/cancer (R. Binder et al., Y. Kato et al., and S. Calderwood et al.); the role of HSPs in the pathogenesis of the autoimmune components of diverse diseases including atherosclerosis (A. Kilic and K. Mandal); systemic lupus erythematosus (H. Shukla and P. Pitha); Behcets disease (J. Shimizu et al.), uveitis (A. Commodaro et al.), and diabetes (C. Blasi et al.); the relationship between infection, particularly M. paratuberculosis, and autoimmunity (C. Dow); the role of small heat-shock protein H11/HspB8 and its homologous proteins in human disease (L. Aurelian et al.); the impact of exercise and metabolic disorders on HSPs (E. Noble and G. Shen); the immunosuppressive activity of HSP70 (P. Stocki and A. Dickinson); and HSP-induced regulatory T cells and their role in control of autoimmunity (E. Brenu et al.). These papers highlight results obtained from studies in animal models as well as patients with autoimmune or metabolic disorders. n nHSPs are highly conserved in nature, and they are also quite immunogenic. These attributes may render these proteins as initiators of immune response as well as targets of autoimmune attack. Foreign (e.g., microbial) HSPs may prime cellular or humoral immune responses that might be cross-reactive with the corresponding self-HSPs or other self-antigens leading to the induction of autoimmunity. “Molecular mimicry” and unveiling of the previously cryptic (hidden) determinants are among the different mechanisms involved in the induction of autoimmunity [3]. Interestingly, foreign-self mimicry may not always be pathogenic; instead, it may be immunoregulatory in nature [13, 14]. Further, defined pathogenic versus regulatory T- or B-cell epitopes have been identified within the same HSP, for example HSP60/65. Accordingly, during the course of autoimmunity, epitope spreading or diversification of response can lead to induction of new T-cell/antibody responses, which in turn may aggravate or downmodulate autoimmunity depending on the antigenic epitopes targeted in the process. Several papers in this special issue discuss the pathogenic role of HSP-induced immunity. The role of particular HSPs in specific autoimmune disorders is highlighted in the papers on atherosclerosis (A. Kilic and K. Mandal), uveitis (A. Commodaro), lupus (H. Shukla and P. Pitha), and Behcets disease (J. Shimizu et al.). A viewpoint on the role of M. paratuberculosis HSP65 in the induction of autoimmunity via molecular mimicry is presented by C. Dow. Two papers in this issue have addressed the relationship between HSPs and metabolic disorders with components of autoimmunity (E. Noble and G. Shen; C. Blasi et al.). n nHSPs are known to play critical roles in innate immunity as well as adaptive immunity. HSPs can activate specific toll-like receptors (TLRs) and influence activation of antigen-presenting cells (APC) and thereby other immune cells including T cells and B cells. These proteins also play a major role in cross-presentation of extracellular antigens such as microbial antigens or tumor antigens in parenchymal cells via the class I pathway resulting in induction of CD8+ cytotoxic T-lymphocyte (CTL) responses [15, 16]. Cross-presentation of HSP-mediated presentation of self-antigens might also play a role in the pathogenesis of autoimmunity. The mechanistic basis of the role of HSPs in cross-presentation of antigens and the impact of this process on immune response in cancer and autoimmunity are elaborated in this issue in papers by S. Calderwood et al., R. Binder et al., and Y. Kato et al. n nHSPs are induced by a variety of stressful stimuli, and they aid in controlling the physical and metabolic integrity of the cells under stress. Metabolic disorders and exercise can induce HSPs and activate other heat-shock factor-1- (HSF-1-) mediated effector pathways. These in turn can enhance the generation of mediators of inflammation. E. Noble and G. Shen discuss in this issue the above-mentioned associations, including their dual role in inflammation. However, C. Blasi et al. report that an effective control of type 2 diabetes was not accompanied by a reduction in serum levels of HSP60 and antibodies to HSP60, while it lowered the levels of the proinflammatory cytokine IL-6. n nAs elaborated above, HSPs are involved in the induction as well as regulation of immune responses. How the same proteins can mediate opposite outcomes is a dilemma for both basic researchers and physician investigators. This dual role of HSPs has been revealed in a wide variety of disorders, including autoimmune diseases and tumors, as well as in immune responses associated with organ transplantation [7, 8, 10, 13, 15, 17, 18]. It is becoming clear that the pro- versus anti-inflammatory activities of HSPs are contextual and affected by multiple factors including the concentration of HSP, the timing of exposure to HSP, and the overall physiopathological milieu at the target site. Another challenge regarding HSPs and immune responses lies in the fact that autoimmunity and tumors present with opposite requirements for control of the disease processes [2, 17]. Autoimmunity involves a breakdown of self-tolerance and induction of anti-self immune responses. Accordingly, an effective control of autoimmunity requires suppression of autoreactive immune responses. On the contrary, tumors survive and grow in the body owing to an ineffective antitumor response in part because of immune-evasive strategies adopted by tumor cells. A deliberate induction of potent antitumor immunity is required to control cancer. HSPs are being exploited differentially to control autoimmunity and cancer. The disparate roles of HSPs in tumor immunity and autoimmunity and the molecular and cellular mechanisms involved therein are discussed here by S. Calderwood et al., R. Binder et al., and L. Aurelian et al. n nHSPs may facilitate regulation of effector responses under appropriate conditions [3, 7–9]. This can be achieved in part via increasing the production of anti-inflammatory cytokines so as to deviate the cytokine balance from a proinflammatory to an anti-inflammatory type. In addition, HSPs may induce different types of regulatory T cells, including CD4+ Tr1 cells that secrete interleukin-10 (IL-10) and CD4+CD25+ Forkhead-box-P3- (Foxp3-) expressing Treg that produce transforming growth factor-b (TGF-β) and IL-10. Treg may also mediate their effect via cell-to-cell contact with the target cells. In this issue, the immunosuppressive activity of HSP is outlined by P. Stocki and A. Dickinson, while HSP-induced regulatory cells are described by E. Brenu et al. n nHSPs are being exploited for immunotherapy of autoimmune diseases and cancer [2, 15, 19, 20]. The current approaches under development or those being tested in clinical trials harness the immunoregulatory properties of HSPs. For example, purified HSPs or their peptides containing defined epitopes are being tested in clinical trials in type 1 diabetes [19] and rheumatoid arthritis [20] patients with the hope of developing immunotherapeutic approaches for these debilitating diseases. The use of HSP-induced regulatory T cells is an emerging area of potential promise in this regard [9]. n nWe hope you enjoy reading the diverse collection of outstanding papers in this special issue.

Monocyte cytokine synthesis in response to extracellular cell stress proteins suggests these proteins exhibit network behaviour

Human peripheral blood monocytes were exposed to single or pairs of cell stress proteins (CSPs), specifically Hsp10, Hsp27, Hsp60 and Hsp70—the former two having anti-inflammatory actions while the latter pair being assumed to be pro-inflammatory in activity. This study was to test if these proteins exhibited any network behaviour. To control for possible lipopolysaccharide contamination, polymyxin B was used. Surprisingly, at concentrations higher than 1xa0μg/ml, polymyxin B itself could induce cytokine synthesis. A number of commercial sources of the molecular chaperones were tested, and marked variations in monocyte cytokine synthesis were found. All four CSPs stimulated the same profile of IL-1/IL-6 synthesis and IL-10/TNF-α synthesis although the kinetics of production of these two pairs of cytokines were very different. A key question was whether extracellular molecular chaperones exhibited network behaviour. To test this, monocytes were cultured with suboptimal concentrations of single CSP and pairs of CSP to look for additive, synergistic or antagonistic cell responses. The major finding was that pairs of molecular chaperones, including chaperones thought to stimulate monocyte cytokine synthesis, could produce significant antagonistic cellular responses. This demonstrates that extracellular CSPs constitute an additional potent layer within the complex cytokine network and furthermore suggests that monocytes have evolved to dampen their immune responses upon exposure to extracellular networks of CSPs—perhaps as a mechanism for protecting cells against detrimental cellular stress responses.

Utilizing confocal laser endomicroscopy for evaluating the adequacy of laparoscopic liver ablation

Laparoscopic liver ablation therapy can be used for the treatment of primary and secondary liver malignancy. The increased incidence of cancer recurrence associated with this approach, has been attributed to the inability of monitoring the extent of ablated liver tissue.

Systemic gene transfer of binding immunoglobulin protein (BiP) prevents disease progression in murine collagen-induced arthritis

Summary

Peripheral blood mononuclear cell proliferative responses to soluble and particulate heat shock protein 65 in health and inflammatory bowel disease.

Abstract.Objectives:The aims of this study were to determine, in peripheral blood mononuclear cells (PBMC), whether particulate antigen triggers (i) an amplified cell proliferative response compared to soluble antigen and (ii) a dysfunctional response in cells derived from patients with chronic inflammation and specifically in those with inflammatory bowel disease (IBD).Subjects:Healthy volunteers (n = 17), inflammatory controls (n = 8) and patients with IBD (n = 17) were recruited from St Thomas’ and Guys’ Hospital, London, UK.Methods:Following optimisation of experimental conditions (0.1–10.0xa0μg/ml antigen), PBMC were stimulated with (i) 10.0xa0μg/ml recombinant soluble heat shock protein 65 (hsp 65) and (ii) 1.0 and 10.0xa0μg/ml hsp 65 conjugated to microparticles (0.5xa0μm diameter). PBMC proliferative responses were measured by 3H-Thymidine incorporation at day 5 and results compared between groups using unpaired t-test.Results:Conjugation to microparticles of low dose hsp 65 significantly increased overall proliferative responses by 2–11 fold compared to soluble antigen alone (p < 0.05). However, no specific PBMC proliferative dysregulation was noted in cells from subjects with IBD.Conclusions:Low dose antigen, in microparticulate form, leads to amplified cell proliferation in primary human cells, as showed previously in cell lines and animal studies. However there is no abnormal proliferative response in cells from subjects with IBD.

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ+ Th1 responses are potentiated in Ptpn22−/− T-cells and in T-cells from mice expressing Ptpn22R619W (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22−/− T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22−/− T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA323-339 pulsed Ptpn22−/− bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.