Inflammatory stress of pancreatic beta cells drives release of extracellular heat-shock protein 90α

A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1β, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity

Proinsulin and heat shock protein 90 as biomarkers of beta-cell stress in the early period after onset of type 1 diabetes

Rapid evaluation of therapies designed to preserve β cells in persons with type 1 diabetes (T1D) is hampered by limited availability of sensitive β-cell health biomarkers. In particular, biomarkers elucidating the presence and degree of β-cell stress are needed. We characterized β-cell secretory activity and stress in 29 new-onset T1D subjects (10.6 ± 3.0 years, 55% male) at diagnosis and then 8.2 ± 1.2 weeks later at first clinic follow-up. We did comparisons with 16 matched healthy controls. We evaluated hemoglobin A1c (HbA1c), β-cell function (random C-peptide [C] and proinsulin [PI]), β-cell stress (PI:C ratio), and the β-cell stress marker heat shock protein (HSP)90 and examined these parameters' relationships with clinical and laboratory characteristics at diagnosis. Mean diagnosis HbA1c was 11.3% (100 mmol/mol) and 7.6% (60 mmol/mol) at follow-up. C-peptide was low at diagnosis (P < 0.001 vs controls) and increased at follow-up (P < 0.001) to comparable with controls. PI did not differ from controls at diagnosis but increased at follow-up (P = 0.003) signifying increased release of PI alongside improved insulin secretion. PI:C ratios and HSP90 concentrations were elevated at both time points. Younger subjects had lower C-peptide and greater PI, PI:C, and HSP90. We also examined islets isolated from prediabetic nonobese diabetic mice and found that HSP90 levels were increased ∼4-fold compared with those in islets isolated from matched CD1 controls, further substantiating HSP90 as a marker of β-cell stress in T1D. Our data indicate that β-cell stress can be assessed using PI:C and HSP90. This stress persists after T1D diagnosis. Therapeutic approaches to reduce β-cell stress in new-onset T1D should be considered

Virus-encoded ectopic CD74 enhances poxvirus vaccine efficacy

Vaccinia virus (VV) has been used globally as a vaccine to eradicate smallpox. Widespread use of this viral vaccine has been tempered in recent years because of its immuno-evasive properties, with restrictions prohibiting VV inoculation of individuals with immune deficiencies or atopic skin diseases. VV infection is known to perturb several pathways for immune recognition including MHC class II (MHCII) and CD1d-restricted antigen presentation. MHCII and CD1d molecules associate with a conserved intracellular chaperone, CD74, also known as invariant chain. Upon VV infection, cellular CD74 levels are significantly reduced in antigen-presenting cells, consistent with the observed destabilization of MHCII molecules. In the current study, the ability of sustained CD74 expression to overcome VV-induced suppression of antigen presentation was investigated. Viral inhibition of MHCII antigen presentation could be partially ameliorated by ectopic expression of CD74 or by infection of cells with a recombinant VV encoding murine CD74 (mCD74-VV). In contrast, virus-induced disruptions in CD1d-mediated antigen presentation persisted even with sustained CD74 expression. Mice immunized with the recombinant mCD74-VV displayed greater protection during VV challenge and more robust anti-VV antibody responses. Together, these observations suggest that recombinant VV vaccines encoding CD74 may be useful tools to improve CD4⁺ T-cell responses to viral and tumour antigens

Allergic airway disease in mice alters T and B cell responses during an acute respiratory poxvirus infection.

Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8(+) T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8(+) T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4(+) T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism

Preexisting AAD exacerbated pulmonary VV infection.

<p>(A) AAD was induced in mice by repeated OVA i.p. sensitizations and respiratory challenges over a course of 19 days. The resulting mice with AAD or control mice were inoculated at day 21 with 10⁴ PFU VV i.t. followed by monitoring for virus-induced pathology. VV titer and weight loss profiles from two separate cohorts of treated animals are shown. (B, D) Virus titers were measured in homogenized lung tissue using a viral plaque assay as described in the methods. Viral persistence and titer were significantly higher in AAD mice 10–12 dpi. The largest difference in virus titer between the VV mice and VV-infected AAD mice was observed at 10 dpi. (C, E) Mice were weighed starting one day after VV infection and the percent weight change was normalized to this day. The kinetics of weight loss after VV infection was not altered by AAD, but the maximal weight loss was significantly increased in AAD+VV mice. All values are represented as mean ± SEM, 4–14 mice per group. (D) Statistical significance was determined by a One-way ANOVA: ^###<i>P</i><0.001 AAD+VV vs. VV. (B, C, E) Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, ***<i>P</i><0.001, ****<i>P</i><0.0001 AAD+VV vs. control; ^#<i>P</i><0.05, ^##<i>P<0.01</i>, ^###<i>P</i><0.001 AAD+VV vs. VV. The following abbreviations are used in all figure legends: VV, vaccinia virus; AAD, allergic airway disease; AAD+VV, allergic airway disease+vaccinia virus. The cohort of mice examined in panels B and C were also used in experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g002" target="_blank">Figures 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g005" target="_blank">5</a>. The cohort of mice examined in panels D and E were also used in experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g006" target="_blank">Figures 6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g007" target="_blank">7</a>.</p

Blocking IL-10R signaling in VV-infected AAD mice resulted in altered disease severity.

<p>VV-infected mice were treated with Rat IgG1 control mAb or αIL-10R blocking mAb at 3 (i.p.), 4 (i.n.) and 6 dpi (i.p.), and these animals were sacrificed on day 9. (A) Lung VV titers and (B) bronchiole inflammation were not altered by IL-10R mAb blockade. Blocking IL-10R signaling increased (C) levels of VV-specific IgG1 in serum, (D) IFNγ protein levels in BAL fluid and (E) the frequency of infiltrating CD4⁺ IFNγ⁺ T cells in the lungs of VV-infected AAD mice. Treatment with an αIL-10R mAb did not alter the infiltration of (F) CD8⁺ IFNγ⁺, (H) CD4⁺ IL-10⁺ or (I) CD8⁺ IL-10⁺ T cells, butincreased recruitment of (J) CD4⁺ PD-1⁺ T cells in the lungs of VV-infected AAD mice. Blocking IL-10R significantly decreased (G) BAL IL-10 protein secretion and significantly increased (K) BAL T cells but not (L) total BAL cells in VV-infected mice with AAD. Statistical significance was determined by a One-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, **<i>P</i><0.01, ****<i>P</i><0.0001 αIL-10R mAb vs. IgG1. Results are expressed as the mean ± SEM for 3–5 mice in each group and are representative of 2 independent experiments.</p

Induction of AAD and pulmonary VV inoculation altered expression of cytokines in the lungs.

<p>Relative expression of gene transcripts in lung tissue was measured using qRT-PCR. Transcripts for pro-allergic cytokines (A) <i>Il13</i>, (B) <i>Il17a</i> and (C) <i>Il5</i>, but not (D) <i>Il6</i>, were increased in AAD and VV-infected AAD mice. Transcripts for (E) <i>Il10</i> and (F) <i>Ifng</i> were elevated in VV-infected mice and VV-infected AAD mice. In VV-infected mice, (G) IL-10 and (H) IFNγ secretion in BAL fluid peaked by 9 dpi, as measured by ELISA. AAD mice inoculated with VV secreted more IL-10 and IFNγ at 9 dpi compared to non-allergic mice infected with VV. Data are expressed as the mean ± SEM for four mice in each group. Data are representative of 2 independent experiments. Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 AAD+VV vs. control; ^#<i>P</i><0.05, ^##<i>P</i><0.01 AAD+VV vs. VV; ⁺⁺⁺<i>P</i><0.001, ⁺⁺⁺⁺<i>P</i><0.0001 AAD+VV vs. AAD.</p

AAD caused increased epithelium disruption and cellular hyperplasia regardless of VV infection.

<p>Murine lung tissue was fixed in 10% formalin and paraffin-embedded sections were stained with PAS/hematoxylin and blindly scored for several pathophysiological parameters using a semi-quantitative scale of 0–3. Mice with AAD had (A) increased bronchiole epithelium disruption (yellow arrow) in the airways, (B) increased perivascular lymphoid hyperplasia (yellow inverted triangle), and (C) increased giant cell pneumonia (red diamond-headed arrow) at 2 and 9 dpi. (D) Goblet cell hyperplasia (yellow box) was significantly increased in AAD+VV mice at 2 dpi, but significantly decreased at 12 dpi compared to AAD mice. (E–G) PAS/hematoxylin-stained slides were digitally imaged with the Aperio Scan Scope CS system at 20× magnification. Multifocal necrotizing pneumonia is evident in the AAD+VV mice at 9 and 12 dpi as diffuse pink staining in the lung parenchyma (red arrow). Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: NS - not significant, ⁺<i>P</i><0.05, ⁺⁺<i>P</i><0.01 ⁺⁺⁺<i>P</i><0.001 AAD+VV vs. AAD.</p

VV-infected AAD mice had increased signs of airway inflammation.

<p>(A) Non-invasive plethysmography was used to assess animal breathing. This analysis revealed increased responses to methacholine challenge (Penh) for control, VV and AAD mice at day 7 post-inoculation or mock treatment. In contrast, AAD mice inoculated with VV had elevated baseline Penh measurements suggesting an altered breathing pattern which was not sensitive to methacholine exposure. (B) Murine lung tissue was fixed in 10% formalin and paraffin-embedded sections were stained with H&E. Lung tissue inflammation was assessed by light microscopy and blindly scored using a semi-quantitative scale of 0–4, with a measure of 0 reflecting no inflammation, and 4 indicative of severe inflammation of peribronchiolar, periarterial and parenchymal spaces. AAD mice with or without VV infection had severe bronchiolar inflammation 2 dpi. VV-infected AAD mice had sustained inflammation through day 12 compared to AAD mice. (C) AAD mice with or without VV infection had elevated inflammatory cell infiltration in the BAL at 2 dpi. VV-infected mice with AAD had prolonged inflammatory cell infiltration in the BAL through 9 dpi. All values represented as mean ± SEM, 4–14 mice per group and representative of 3 independent experiments. Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 AAD+VV vs. control; ^#<i>P</i><0.05, AAD+VV vs. VV. ⁺⁺⁺<i>P</i><0.001 AAD+VV vs. AAD.</p