A Fiber-Rich Diet and Radiation-Induced Injury in the Murine Intestinal Mucosa

Dietary fiber is considered a strong intestinal protector, but we do not know whether dietary fiber protects against the long-lasting mucosal damage caused by ionizing radiation. To evaluate whether a fiber-rich diet can ameliorate the long-lasting pathophysiological hallmarks of the irradiated mucosa, C57BL/6J mice on a fiber-rich bioprocessed oat bran diet or a fiber-free diet received 32 Gray in four fractions to the distal colorectum using a linear accelerator and continued on the diets for one, six or 18 weeks. We quantified degenerating crypts, crypt fission, cell proliferation, crypt survival, macrophage density and bacterial infiltration. Crypt loss through crypt degeneration only occurred in the irradiated mice. Initially, it was most frequent in the fiber-deprived group but declined to levels similar to the fiber-consuming group by 18 weeks. The fiber-consuming group had a fast response to irradiation, with crypt fission for growth or healing peaking already at one week post-irradiation, while crypt fission in the fiber-deprived group peaked at six weeks. A fiber-rich diet allowed for a more intense crypt cell proliferation, but the recovery of crypts was eventually lost by 18 weeks. Bacterial infiltration was a late phenomenon, evident in the fiber-deprived animals and intensified manyfold after irradiation. Bacterial infiltration also coincided with a specific pro-inflammatory serum cytokine profile. In contrast, mice on a fiber-rich diet were completely protected from irradiation-induced bacterial infiltration and exhibited a similar serum cytokine profile as sham-irradiated mice on a fiber-rich diet. Our findings provide ample evidence that dietary fiber consumption modifies the onset, timing and intensity of radiation-induced pathophysiological processes in the intestinal mucosa. However, we need more knowledge, not least from clinical studies, before this finding can be introduced to a new and refined clinical practice.

Analysis of Macrophages and Peptidergic Fibers in the Skin of Patients With Painful Diabetic Polyneuropathy

Background and ObjectivesThe mechanisms of pain in patients with diabetic polyneuropathy are unknown. Studies have suggested a role of inflammation and increased neuropeptides peripherally in pain generation. This study examined the possible skin markers of painful diabetic polyneuropathy (P-DPN): macrophages, substance P (SP), and calcitonin gene-related peptide (CGRP).MethodsThe participants were included from a large Danish cross-sectional clinical study of type 2 diabetes. We diagnosed definite diabetic polyneuropathy using the Toronto criteria and used the Neuropathic Pain Special Interest Group classification for defining P-DPN. We included 60 skin biopsies from patients with diabetic polyneuropathy—30 with P-DPN and 30 with nonpainful diabetic polyneuropathy (NP-DPN)—and 30 biopsies from healthy controls of similar age and sex. The biopsies were stained using PGP 9.5, IbA1, and SP and CGRP primary markers.ResultsThere was increased macrophage density in patients with P-DPN (8.0%) compared with that in patients with NP-DPN (5.1%, p < 0.001), and there was increased macrophage density in patients with NP-DPN (5.1%) compared with that in healthy controls (3.1%, p < 0.001). When controlling for neuropathy severity, body mass index, age, and sex, there was still a difference in macrophage density between patients with P-DPN and patients with NP-DPN. Patients with P-DPN had higher median nerve fiber length density (274.5 and 155 mm−2 for SP and CGRP, respectively) compared with patients with NP-DPN (176 and 121 mm−2 for SP and CGRP, respectively, p = 0.009 and 0.04) and healthy controls (185.5 and 121.5 mm−2 for SP and CGRP, respectively), whereas there was no difference between patients with NP-DPN and controls without diabetes (p = 0.64 and 0.49, respectively). The difference between P-DPN and NP-DPN for SP and CGRP was significant only in female patients, although a trend was seen in male patients.DiscussionThe findings point to a possible involvement of the innate immune system in the pathogenesis of neuropathic pain in patients with DPN, although markers of activated macrophages were not measured in this study.

Regulation of CNS Lymphoma Progression By the Myeloid Microenvironment

;'Insights into the molecular and immunologic pathogenesis of primary CNS lymphomas are essential for meaningful progress in therapy. Tumor-associated macrophages represent the dominant infiltrating leukocyte and there are few established insights into their phenotypes and role in this disease. While upregulation of Th2 cytokines IL-4 and IL-10 in the microenvironment has been demonstrated, the relative roles of M1 and M2 macrophages in contributing to CNS lymphoma pathogenesis has not been elucidated. To date, there is also no information regarding the relative contributions of brain resident microglia and infiltrating macrophages and their interactions with lymphoma. Additional key questions include the identification of factors that mediate both immune cell chemotaxis in CNS lymphomas, as well as the relationship between myeloid cell infiltration and T-cell mediated immune surveillance and immunosuppression. We combined analyses of clinical specimens and mechanistic studies using preclinical in vivo models and show evidence that infiltrating tumor-associated macrophages, derived from monocyte precursors, have a critical role in attenuating CNS lymphoma progression. Immunohistochemical analysis of the density and morphologic features of CD68+ tumor-associated macrophages in 62 diagnostic specimens of immunocompetent PCNSL demonstrated that smaller macrophage size and lower macrophage density correlated with significantly shorter OS. Evaluation of CD68 immunoreactivity using image analysis software (ImageJ) confirmed the heterogeneity of macrophage size and infiltrative density in PCNSL. A multivariate Cox model including age, IELSG score, receipt of consolidation and/or maintenance therapy demonstrated that tumor-associated macrophage density (both count and area) was a significant, independent predictor of favorable PFS and OS and that larger macrophage size a significant, independent predictor of OS in PCNSL treated with standard MTX-based induction (predominantly MTX, temozolomide, rituximab). Using a variety of syngeneic and non-syngeneic preclinical models, including patient-derived CNS lymphoma cells, as well as diagnostic clinical specimens, we characterized the phenotype of tumor-associated macrophages in PCNSL. Using flow-cytometry, we demonstrated that while CD45 high tumor-associated macrophages exhibit strong expression of the canonical M2 marker CD206, a scavenger receptor, these also displayed high co-expression of iNOS and MHC II, markers of classically-activated M1 macrophages. Pharmacologic inhibition of the CSF-1 receptor led to accelerated CNS lymphoma progression, attenuated T-cell infiltration and blocked rituximab efficacy. A flow-cytometric assay of phagocytosis, using Raji lymphoma transduced to express mCherry, demonstrated that infiltrating CD206+ macrophages are the dominant mediator of lymphoma phagocytosis. We applied 2P intravital imaging of a CNS lymphoma model using Cx3cr1GFP/+:Ccr2RFP/+ myeloid cell dual reporter mice and transcriptional studies to define the time-dependent infiltration and phenotypic changes in tumor-associated macrophages and microglia that correlate with disease progression. Using IFN-γ -/- mice we identified a critical role for IFN-γ in the regulation of CNS lymphoma, in the presence and absence of T-cells. We identified IFN-γ-regulated genes in tumor-associated macrophages that may contribute to direct lymphoma cytotoxicity as well as stimulation of T-cell chemotaxis and antigen processing, including TAP1 and TAP2. By IHC, we confirmed TAP1 expression in a subset of diagnostic specimens of PCNSL and determined, using Cox multivariate model, that strong TAP1 correlated with improved PFS (p&lt;0.0006). Notably, independent of receipt of maintenance therapy, TAP1 also correlated with improved PFS in 38 patients that received only MTX-based induction, without dose-intensive chemotherapy consolidation. (Figure 1) Our results support a direct, immune-editing role for monocyte-derived macrophages in the regulation of CNS lymphoma progression, via several mechanisms, including antigenic processing and cross-presentation. We suggest that tumor-associated CD68 and TAP1 (and TAP2) be evaluated further as candidate biomarkers for risk stratification in PCNSL, particularly in trials that involve targeted immunotherapy. Supported by NCI and LLS. Figure 1 Figure 1. Disclosures Rubenstein: Kymera: Research Funding.

Abstract MP255: Intercellular Model Predicts Macrophage Signaling As A Driver Of Cardiac Fibroblast Response Post Myocardial Infarction

Introduction: Post-myocardial infarction (MI), cardiac fibroblasts and macrophages work together to regulate tissue homeostasis and infarct repair. Macrophage-fibroblast interactions in healthy tissue are stable and resistant to perturbations. However, this robustness post-MI has not been assessed. This study designs and implements an intercellular communication model of macrophage-fibroblast crosstalk to determine drivers of infarct repair. Methods: An ordinary differential equation model of post-MI cellular dynamics was developed ( Figure 1A ). Model inputs are time courses of cardiomyocytes, neutrophils, and monocytes. These cells, along with simulated macrophages and fibroblasts, secrete chemokines and cytokines which directed cell proliferation, removal, and chemical secretion. The outputs are macrophage and fibroblast densities, secreted factor dynamics, and produced collagen. Model validation was done using published data in post-MI mice. A sensitivity analysis was conducted by knocking down individual parameters to identify key drivers of fibroblast collagen production. Results: The simulated trends matched the validation time courses. Of the 28 validation relationships, 12 were input-output, 7 were knockouts, and 9 were inhibitor relationships. The validation passed at 78.5%; the 6 failed validations were due to the independent nature of the input curves. Sensitivity analysis ( Figure 1B ) identified macrophage differentiation rate, removal rate, and transforming growth factor-beta (TGFB) secretion rate as pro-fibrotic. Prolonged exposure to TGFB and granulocyte-macrophage colony stimulating factor was anti-fibrotic. Several clustered parameters differentially regulated macrophages and collagen production. Conclusions: The multicellular model identified macrophage density as a pro-fibrotic driver in the healing infarct. Differential regulation of macrophages and collagen production was predicted by the model.

Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.

Synovial Immunohistological Biomarkers of the Classification of Undifferentiated Arthritis Evolving to Rheumatoid or Psoriatic Arthritis

Background: Undifferentiated arthritis (UA) is defined as an inflammatory arthritis that does not fulfill criteria for a definite diagnosis. Delay in reaching a specific diagnostic and therapy may lead to impaired functional outcomes. Our aim was to identify synovial biomarkers associated with definitive diagnostic classification in patients with UA.Methods: DMARD-naïve UA patients with available initial synovial tissue (ST) and a final diagnosis of rheumatoid arthritis (RA) or psoriatic arthritis (PsA) during follow-up were included and compared with patients with well-defined disease (RA or PsA). Clinical, arthroscopic, and pathological data were compared between groups. Pathology included quantitative immunohistochemical (IHC) analysis of cell types and human interferon-regulated MxA. Principal component analysis (PCA) was performed to extract disease patterns.Results: One hundred and five patients were included: 31 patients with DMARD-naïve UA (19 evolving to RA and 12 to PsA during a median follow up of 7 years), 39 with established RA, and 35 with established PsA. ST from the UA group showed higher macrophage density compared with the established RA and PsA groups. Patients with UA evolving to RA (UA-RA) showed higher MxA expression and CD3+ T-cell density compared with established RA. UA patients evolving to PsA (UA-PsA) showed increased vascularity and lining synovial fibroblast density compared with established PsA. Synovitis of UA-PsA patients showed more mast cells and lining fibroblasts compared with UA-RA. No between-group differences in local or systemic inflammation markers were found.Conclusions: Our results show differences in the cellular composition of UA synovium compared with RA and PsA, with higher density of the cellular infiltrate in the UA groups. Initial expression of the interferon inducible gene MxA could be a biomarker of progression to RA, while higher mast cell and fibroblastic density may be associated with PsA progression.

CMTM6 expression in M2 macrophages is a potential predictor of PD-1/PD-L1 inhibitor response in colorectal cancer

Background CMTM6 is a novel key regulator of PD-L1. High expression of both CMTM6 and PD-L1 may predict the benefit of PD-1 axis blockade in lung cancer. We aimed to investigate the expression pattern of CMTM6 between mismatch repair-defective (dMMR) and mismatch repair-proficient (pMMR) colorectal cancer (CRC) tissues and assess its correlation with the response to PD-1/PD-L1 pathway blockade. Methods Immunohistochemistry (IHC) was used to analyze CMTM6 and PD-L1 expression and immune cell density in dMMR/pMMR CRC. Quantitative multiplex immunofluorescence (IF) was performed to detect CMTM6, PD-L1, CD4, CD8, CD68 and CD163 expression in CRC patients treated with PD-1/PD-L1 inhibitors. Result IHC analysis showed that CMTM6 and PD-L1 were both expressed in tumor cells (TCs) and invasion front immune cells (ICs). CMTM6 and PD-L1 expression and CD4+, CD8+, CD68+ or CD163+ cell density were significantly higher in dMMR CRC patients than in pMMR CRC patients. CMTM6 expression was positively correlated with PD-L1 expression and CD163+ M2 macrophage density in dMMR CRC. IF analysis showed that the coexpression rate of CMTM6/PD-L1 and the expression rate of CMTM6 in CD8+ T cells and CD163+ M2 macrophages were significantly increased in the group that exhibited clinical benefit. CMTM6 expression in M2 macrophages was identified as the best biomarker for predicting the responsiveness to PD-1/PD-L1 inhibitors. Conclusions CMTM6 expression in M2 macrophages may predict the PD-1/PD-L1 inhibitor response rate in CRC patients more accurately than dMMR/microsatellite instability-high (MSI-H) status. It can also identify pMMR CRC patients who could benefit from PD-1/PD-L1 inhibitors.

GM-CSF Expression and Macrophage Polarization in Joints of Undifferentiated Arthritis Patients Evolving to Rheumatoid Arthritis or Psoriatic Arthritis

Background and AimsGM-CSF-dependent macrophage polarization has been demonstrated in rheumatoid arthritis (RA). Our aim was to seek diagnostic/prognostic biomarkers for undifferentiated arthritis (UA) by analyzing GM-CSF expression and source, macrophage polarization and density in joints of patients with UA evolving to RA or PsA compared with established RA or PsA, respectively.MethodsSynovial tissue (ST) from patients with UA evolving to RA (UA&gt;RA, n=8), PsA (UA&gt;PsA, n=9), persistent UA (UA, n=16), established RA (n=12) and PsA (n=10), and healthy controls (n=6), were analyzed. Cell source and quantitative expression of GM-CSF and proteins associated with pro-inflammatory (GM-CSF-driven) and anti-inflammatory (M-CSF-driven) macrophage polarization (activin A, TNFα, MMP12, and CD209, respectively) were assessed in ST CD163+ macrophages by multicolor immunofluorescence. GM-CSF and activin A levels were also quantified in paired synovial fluid samples. CD163+ macrophage density was determined in all groups by immunofluorescence.ResultsSynovial stromal cells (FAP+ CD90+ fibroblast, CD90+ endothelial cells) and CD163+ sublining macrophages were the sources of GM-CSF. ST CD163+ macrophages from all groups expressed pro-inflammatory polarization markers (activin A, TNFα, and MMP12). Expression of the M-CSF-dependent anti-inflammatory marker CD209 identified two macrophage subsets (CD163+ CD209high and CD163+ CD209low/-). CD209+ macrophages were more abundant in ST from healthy controls and PsA patients, although both macrophage subtypes showed similar levels of pro-inflammatory markers in all groups. In paired synovial fluid samples, activin A was detected in all patients, with higher levels in UA&gt;RA and RA, while GM-CSF was infrequently detected. ST CD163+ macrophage density was comparable between UA&gt;RA and UA&gt;PsA patients, but significantly higher than in persistent UA.ConclusionsGM-CSF is highly expressed by sublining CD90+ FAP+ synovial fibroblasts, CD90+ activated endothelium and CD163+ macrophages in different types of arthritis. The polarization state of ST macrophages was similar in all UA and established arthritis groups, with a predominance of pro-inflammatory GM-CSF-associated markers. CD163+ macrophage density was significantly higher in the UA phases of RA and PsA compared with persistent UA. Taken together, our findings support the idea that GM-CSF is a strong driver of macrophage polarization and a potential therapeutic target not only in RA but also in PsA and all types of UA.

Chronic exposure to IL-6 induces a desensitized phenotype of the microglia

Background When the homeostasis of the central nervous system (CNS) is altered, microglial cells become activated displaying a wide range of phenotypes that depend on the specific site, the nature of the activator, and particularly the microenvironment generated by the lesion. Cytokines are important signals involved in the modulation of the molecular microenvironment and hence play a pivotal role in orchestrating microglial activation. Among them, interleukin-6 (IL-6) is a pleiotropic cytokine described in a wide range of pathological conditions as a potent inducer and modulator of microglial activation, but with contradictory results regarding its detrimental or beneficial functions. The objective of the present study was to evaluate the effects of chronic IL-6 production on the immune response associated with CNS-axonal anterograde degeneration. Methods The perforant pathway transection (PPT) paradigm was used in transgenic mice with astrocyte-targeted IL6-production (GFAP-IL6Tg). At 2, 3, 7, 14, and 21 days post-lesion, the hippocampal areas were processed for immunohistochemistry, flow cytometry, and protein microarray. Results An increase in the microglia/macrophage density was observed in GFAP-IL6Tg animals in non-lesion conditions and at later time-points after PPT, associated with higher microglial proliferation and a major monocyte/macrophage cell infiltration. Besides, in homeostasis, GFAP-IL6Tg showed an environment usually linked with an innate immune response, with more perivascular CD11b+/CD45high/MHCII+/CD86+ macrophages, higher T cell infiltration, and higher IL-10, IL-13, IL-17, and IL-6 production. After PPT, WT animals show a change in microglia phenotype expressing MHCII and co-stimulatory molecules, whereas transgenic mice lack this shift. This lack of response in the GFAP-IL6Tg was associated with lower axonal sprouting. Conclusions Chronic exposure to IL-6 induces a desensitized phenotype of the microglia.

Suppression of Metastatic Melanoma Growth in Lung by Modulated Electro-Hyperthermia Monitored by a Minimally Invasive Heat Stress Testing Approach in Mice

Modulated electro-hyperthermia (mEHT) is a novel complementary therapy in oncology which is based on the higher conductivity and permittivity of cancerous tissues due to their enhanced glycolytic activity and ionic content compared to healthy normal tissues. We aimed to evaluate the potential of mEHT, inducing local hyperthermia, in the treatment of pulmonary metastatic melanoma. Our primary objective was the optimization of mEHT for targeted lung treatment as well as to identify the mechanism of its potential anti-tumor effect in the B16F10 mouse melanoma pulmonary metastases model while investigating the potential treatment-related side effects of mEHT on normal lung tissue. Repeated treatment of tumor-bearing lungs with mEHT induced significant anti-tumor effects as demonstrated by the lower number of tumor nodules and the downregulation of Ki67 expression in treated tumor cells. mEHT treatment provoked significant DNA double-strand breaks indicated by the increased expression of phosphorylated H2AX protein in treated tumors, although treatment-induced elevation of cleaved/activated caspase-3 expression was insignificant, suggesting the minimal role of apoptosis in this process. The mEHT-related significant increase in p21waf1 positive tumor cells suggested that p21waf1-mediated cell cycle arrest plays an important role in the anti-tumor effect of mEHT on melanoma metastases. Significantly increased CD3+, CD8+ T-lymphocytes, and F4/80+CD11b+ macrophage density in the whole lung and tumor of treated animals emphasizes the mobilizing capability of mEHT on immune cells. In conclusion, mEHT can reduce the growth potential of melanoma, thus offering itself as a complementary therapeutic option to chemo- and/or radiotherapy.