Cysteine-type cathepsins promote the effector phase of acute cutaneous delayed-type hypersensitivity reactions.

Cysteine-type cathepsins such as cathepsin B are involved in various steps of inflammatory processes such as antigen processing and angiogenesis. Here, we uncovered the role of cysteine-type cathepsins in the effector phase of T cell-driven cutaneous delayed-type hypersensitivity reactions (DTHR) and the implication of this role on therapeutic cathepsin B-specific inhibition. Methods: Wild-type, cathepsin B-deficient (Ctsb-/-) and cathepsin Z-deficient (Ctsz-/-) mice were sensitized with 2,4,6-trinitrochlorobenzene (TNCB) on the abdomen and challenged with TNCB on the right ear to induce acute and chronic cutaneous DTHR. The severity of cutaneous DTHR was assessed by evaluating ear swelling responses and histopathology. We performed fluorescence microscopy on tissue from inflamed ears and lymph nodes of wild-type mice, as well as on biopsies from psoriasis patients, focusing on cathepsin B expression by T cells, B cells, macrophages, dendritic cells and NK cells. Cathepsin activity was determined noninvasively by optical imaging employing protease-activated substrate-like probes. Cathepsin expression and activity were validated ex vivo by covalent active site labeling of proteases and Western blotting. Results: Noninvasive in vivo optical imaging revealed strong cysteine-type cathepsin activity in inflamed ears and draining lymph nodes in acute and chronic cutaneous DTHR. In inflamed ears and draining lymph nodes, cathepsin B was expressed by neutrophils, dendritic cells, macrophages, B, T and natural killer (NK) cells. Similar expression patterns were found in psoriatic plaques of patients. The biochemical methods confirmed active cathepsin B in tissues of mice with cutaneous DTHR. Topically applied cathepsin B inhibitors significantly reduced ear swelling in acute but not chronic DTHR. Compared with wild-type mice, Ctsb-/- mice exhibited an enhanced ear swelling response during acute DTHR despite a lack of cathepsin B expression. Cathepsin Z, a protease closely related to cathepsin B, revealed compensatory expression in inflamed ears of Ctsb-/- mice, while cathepsin B expression was reciprocally elevated in Ctsz-/- mice. Conclusion: Cathepsin B is actively involved in the effector phase of acute cutaneous DTHR. Thus, topically applied cathepsin B inhibitors might effectively limit DTHR such as contact dermatitis or psoriasis. However, the cathepsin B and Z knockout mouse experiments suggested a complementary role for these two cysteine-type proteases.

The administration route of tumor-antigen-specific T-helper cells differentially modulates the tumor microenvironment and senescence.

Cancer treatment with adoptively transferred tumor-associated antigen-specific CD4+ T-helper cells is a promising immunotherapeutic approach. In the pancreatic cancer model RIP-Tag2, the intraperitoneal (i.p.) application of Tag-specific TH1 cells exhibited a profound antitumoral efficiency. We investigated, whether an intravenous (i.v.) application of Tag-TH1 cells induces an equivalent therapeutic effect. Adoptively transferred fluorescent Tag-TH1 cells revealed a pronounced homing to the tumors after either i.p. or i.v. transfer, and both routes induced an almost equivalent therapeutic effect as demonstrated by magnetic resonance imaging, blood glucose level course and histology. The i.v. administration of Tag-TH1 cells induced p16INK4-positive/Ki67-negative tumor senescence more efficiently than i.p. administration. Both routes replenish host CD4+ T cells by transferred T cells and recruitment of B and dendritic cells to the tumors while reducing CD8+ T cells and depleting macrophages. Both administration routes efficiently induced a similar antitumoral efficiency despite the pronounced senescence induction after i.v. administration. Thus, a combinatory i.v./i.p. injection of therapeutic cells might overcome limitations of the individual routes and improve therapeutic efficacy in solid tumors.

Quantification of ß-Amyloidosis and rCBF with Dedicated PET, 7 T MR Imaging, and High-Resolution Microscopic MR Imaging at 16.4 T in APP23 Mice.

UNLABELLED: We present a combined PET/7 T MR imaging and 16.4 T microscopic MR imaging dual-modality imaging approach enabling quantification of the amyloid load at high sensitivity and high resolution, and of regional cerebral blood flow (rCBF) in the brain of transgenic APP23 mice. Moreover, we demonstrate a novel, voxel-based correlative data analysis method for in-depth evaluation of amyloid PET and rCBF data. METHODS: We injected 11C-Pittsburgh compound B (PIB) intravenously in transgenic and control APP23 mice and performed dynamic PET measurements. rCBF data were recorded with a flow-sensitive alternating inversion recovery approach at 7 T. Subsequently, the animals were sacrificed and their brains harvested for ex vivo microscopic MR imaging at 16.4 T with a T2*-weighted gradient-echo sequence at 30-µm spatial resolution. Additionally, correlative amyloid histology was performed. The 11C-PIB PET data were quantified to nondisplaceable binding potentials (BPND) using the Logan graphical analysis; flow-sensitive alternating inversion recovery data were quantified with a simplified version of the Bloch equation. RESULTS: Amyloid load assessed by both 11C-PIB PET and amyloid histology was highest in the frontal cortex of transgenic mice (11C-PIB BPND: 0.93±0.08; amyloid histology: 15.1%±1.5%), followed by the temporoparietal cortex (11C-PIB BPND: 0.75±0.08; amyloid histology: 13.9%±0.7%) and the hippocampus (11C-PIB BPND: 0.71±0.09; amyloid histology: 9.2%±0.9%), and was lowest in the thalamus (11C-PIB BPND: 0.40±0.07; amyloid histology: 6.6%±0.6%). However, 11C-PIB BPND and amyloid histology linearly correlated (R2=0.82, P<0.05) and were significantly higher in transgenic animals (P<0.01). Similarly, microscopic MR imaging allowed quantifying the amyloid load, in addition to the detection of substructures within single amyloid plaques correlating with amyloid deposition density and the measurement of hippocampal atrophy. Finally, we found an inverse relationship between 11C-PIB BPND and rCBF MR imaging in the voxel-based analysis that was absent in control mice (slopetg: -0.11±0.03; slopeco: 0.004±0.005; P=0.014). CONCLUSION: Our dual-modality imaging approach using 11C-PIB PET/7 T MR imaging and 16.4 T microscopic MR imaging allowed amyloid-load quantification with high sensitivity and high resolution, the identification of substructures within single amyloid plaques, and the quantification of rCBF.

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle.

METHODS: CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O2). We performed 10 min static PET scans 1 h, 2 h and 3 h after [18F]FAZA injection and calculated the [18F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO2 probe in the tumor as well as in the gastrocnemius muscle to measure the pO2 and perfusion. RESULTS: Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [18F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O2: 4.4 vs. IF-O2: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [18F]FAZA uptake in the carcinomas. Invasive pO2 probe measurements yielded enhanced intra-tumoral pO2 values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O2 9.73 mmHg, IF-O2: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003). CONCLUSION: [18F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [18F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO2-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.

64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET.

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific (64)Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied (64)Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.

In vivo optical imaging of matrix metalloproteinase activity detects acute and chronic contact hypersensitivity reactions and enables monitoring of the antiinflammatory effects of N-acetylcysteine.

The aim of this study was to determine whether the severity of contact hypersensitivity reactions (CHSRs) can be observed by noninvasive in vivo optical imaging of matrix metalloproteinase (MMP) activity and whether this is an appropriate tool for monitoring an antiinflammatory effect. Acute and chronic CHSRs were elicited by application of a 1% trinitrochlorobenzene (TNCB) solution for up to five times on the right ear of TNCB-sensitized mice. N-Acetylcysteine (NAC)-treated and sham-treated mice were monitored by measuring ear swelling and optical imaging of MMP activity. In addition, we performed hematoxylin-eosin staining and CD31 immunohistochemistry for histopathologic analysis of the antiinflammatory effects of NAC. The ear thickness and the MMP activity increased in line with the increasing severity of the CHSR. MMP activity was enhanced 2.5- to 2.7-fold during acute CHSR and 3.1- to 4.1-fold during chronic CHSR. NAC suppressed ear swelling and MMP signal intensity in mice with acute and chronic CHSR. During chronic CHSR, the vessel density was significantly reduced in ear sections derived from NAC-treated compared to sham-treated mice. In vivo optical imaging of MMP activity measures acute and chronic CHSR and is useful to monitor antiinflammatory effects.

In vivo tracking of Th1 cells by PET reveals quantitative and temporal distribution and specific homing in lymphatic tissue.

UNLABELLED: Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. METHODS: We developed protocols that minimize the inhibitory effects of (64)Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((64)Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ-producing CD4(+) T (Th1) cells with 0.7-2.2 MBq of (64)Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular (64)Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10(7 64)Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of (64)Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10(7 64)Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. RESULTS: PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, (64)Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered (64)Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of (64)Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm(3)]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity-diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm(3)). As expected, (64)Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm(3)) when compared with phosphate-buffered saline-challenged animals (4.6 ± 0.5 %ID/cm(3)). CONCLUSION: Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for (64)Cu-PTSM-labeled T cells in clinical trials and novel therapy concepts focusing on T-cell-based immunotherapies of autoimmune diseases or cancer.

Multimodal elucidation of choline metabolism in a murine glioma model using magnetic resonance spectroscopy and 11C-choline positron emission tomography.

The metabolites, transporters, and enzymes involved in choline metabolism are regarded as biomarkers for disease progression in a variety of cancers, but their in vivo detection is not ideal. Both magnetic resonance spectroscopy [MRS using chemical shift imaging (CSI) total choline (tCho)] and C-choline positron emission tomography (PET) can probe this pathway, but they have not been compared side by side. In this study, we used the spontaneous murine astrocytoma model SMA560 injected intracranially into syngeneic VM/Dk mice, analyzing animals at various postimplantation time points using dynamic microPET imaging and CSI MRS. We observed an increase in tumor volume and C-choline uptake between days 5 and 18. Similarly, tCho levels decreased at days 5 to 18. We found a negative correlation between the tCho and PET results in the tumor and a positive correlation between the tCho tumor-to-brain ratio and choline uptake in the tumor. PCR results confirmed expected increases in expression levels for most of the transporters and enzymes. Using MRS quantification, a good agreement was found between CSI and C-choline PET data, whereas a negative correlation occurred when CSI was not referenced. Thus, C-choline PET and MRS methods seemed to be complementary in strengths. While advancing tumor proliferation caused an increasing C-choline uptake, gliosis and inflammation potentially accounted for a high peritumoral tCho signal in CSI, as supported by histology and secondary ion mass spectrometry imaging. Our findings provide definitive evidence of the use of MRS, CSI, and PET for imaging tumors in vivo.

Significant impact of different oxygen breathing conditions on noninvasive in vivo tumor-hypoxia imaging using [¹8F]-fluoro-azomycinarabino-furanoside ([¹8F]FAZA).

BACKGROUND: [18F]FAZA is a PET biomarker with great potential for imaging tumor hypoxia. Aim of our study was to compare [18F]FAZA uptake in mice with subcutaneous exogenous CT26 colon carcinomas and endogenous polyoma middle-T (PyV-mT) mammary carcinomas and to analyze the influence of different breathing protocols in CT26 colon carcinomas as well as the reversibility or irreversibility of [18F]FAZA uptake. METHODS: We injected subcutaneous CT26 colon carcinoma or polyomavirus middle-T (PyV-mT) mammary carcinoma-bearing mice intravenously with18F-FAZA and performed PET scans 1-3 h post injection (p.i.). To analyze the impact of oxygen supply in CT26 carcinomas we used three different breathing protocols: (P0) air; (P1) 100% oxygen 1 h prior injection until 3 h p.i.; (P2) 100% oxygen breathing starting 2 min prior tracer injection until 1 h p.i. and during the PET scans; mice were breathing air between the 2 h and 3 h 10 min static scans. Normalized PET images were analyzed by using defined regions of interest. Finally, some mice were dissected for pimonidazole immunohistochemistry. RESULTS: There was no difference in18F-FAZA uptake 1-3 h p.i. between the two carcinoma types (CT26: 1.58 ± 0.45%ID/cc; PyV-mT: 1.47 ± 0.89%ID/cc, 1 h p.i., tumor size < 0.5 cm3). We measured a significant tracer clearance, which was more pronounced in muscle tissue (P0). The [18F]FAZA tumor-to-muscle-ratios in CT26 colon carcinoma-bearing mice 2 h and 3 h, but not 1 h p.i. were significantly higher when the mice breathed air (P0: 3.56 ± 0.55, 3 h) compared to the oxygen breathing protocols (P1: 2.45 ± 0.58; P2: 2.77 ± 0.42, 3 h). Surprisingly, the breathing protocols P1 and P2 showed no significant differences in T/M ratios, thus indicating that the crucial [18F]FAZA uptake phase is during the first hour after [18F]FAZA injection. Importantly, the muscle clearance was not affected by the different oxygen breathing conditions while the tumor clearance was lower when mice were breathing air. CONCLUSION: Exogenous CT26 colon carcinomas and endogenous polyoma middle-T (PyV-mT) mammary carcinomas showed no differences in [18F]FAZA uptake 1-3 h p.i. Our analysis using various breathing protocols with air (P0) and with pure oxygen (P1, P2) clearly indicate that [18F]FAZA is an appropriate PET biomarker for in vivo analysis of hypoxia revealing an enhanced tracer uptake in tumors with reduced oxygen supply. [18F]FAZA uptake was independent of tumor-type.

Direct crosstalk between mast cell-TNF and TNFR1-expressing endothelia mediates local tissue inflammation.

Signaling through tumor necrosis factor receptor 1 (TNFR1) controls bacterial infections and the induction of inflammatory Th1 cell-mediated autoimmune diseases. By dissecting Th1 cell-mediated delayed-type hypersensitivity responses (DTHRs) into single steps, we localized a central defect to the missing TNFR1 expression by endothelial cells (ECs). Adoptive transfer and mast cell knockin experiments into Kit(W)/Kit(W-v), TNF(-/-), and TNFR1(-/-) mice showed that the signaling defect exclusively affects mast cell-EC interactions but not T cells or antigen-presenting cells. As a consequence, TNFR1(-/-) mice had strongly reduced mRNA and protein expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during DTHR elicitation. In consequence, intravital fluorescence microscopy revealed up to 80% reduction of leukocyte rolling and firm adhesion in TNFR1(-/-) mice. As substitution of TNF(-/-) mice with TNF-producing mast cells fully restored DTHR in these mice, signaling of mast cell-derived TNF through TNFR1-expressing ECs is essential for the recruitment of leukocytes into sites of inflammation.