Unfolding innate mechanisms in the cancer microenvironment: The emerging role of the mesenchyme.

Innate mechanisms in the tumor stroma play a crucial role both in the initial rejection of tumors and in cancer promotion. Here, we provide a concise overview of the innate system in cancer and recent advances in the field, including the activation and functions of innate immune cells and the emerging innate properties and modulatory roles of the fibroblastic mesenchyme. Novel insights into the diverse identities and functions of the innate immune and mesenchymal cells in the microenvironment of tumors should lead to improved anticancer therapies.

Immunologic and Non-Immunologic Mechanisms Leading to Airway Remodeling in Asthma.

Asthma increases worldwide without any definite reason and patient numbers double every 10 years. Drugs used for asthma therapy relax the muscles and reduce inflammation, but none of them inhibited airway wall remodeling in clinical studies. Airway wall remodeling can either be induced through pro-inflammatory cytokines released by immune cells, or direct binding of IgE to smooth muscle cells, or non-immunological stimuli. Increasing evidence suggests that airway wall remodeling is initiated early in life by epigenetic events that lead to cell type specific pathologies, and modulate the interaction between epithelial and sub-epithelial cells. Animal models are only available for remodeling in allergic asthma, but none for non-allergic asthma. In human asthma, the mechanisms leading to airway wall remodeling are not well understood. In order to improve the understanding of this asthma pathology, the definition of "remodeling" needs to be better specified as it summarizes a wide range of tissue structural changes. Second, it needs to be assessed if specific remodeling patterns occur in specific asthma pheno- or endo-types. Third, the interaction of the immune cells with tissue forming cells needs to be assessed in both directions; e.g., do immune cells always stimulate tissue cells or are inflamed tissue cells calling immune cells to the rescue? This review aims to provide an overview on immunologic and non-immunologic mechanisms controlling airway wall remodeling in asthma.

Nodular typhlocolitis, heterakiasis, and mesenchymal neoplasia in a ring-necked pheasant ( Phasianus colchicus) with immunohistochemical characterization of visceral metastases.

A 9-y-old, male ring-necked pheasant ( Phasianus colchicus) was autopsied following euthanasia because of acute distress, recumbency, and dyspnea. The bird had experienced a protracted period of neuromuscular disease localized to the left sciatic nerve. Gross and histologic examination of the large intestine revealed intramural nodules predominantly comprised of atypical, whorling spindle cells with small cores of granulomatous inflammation centered on cross-sections of immature adult nematodes. The body structures of these metazoan organisms and clinical disease manifestation are consistent with Heterakis isolonche infection. Nodular spindle cell proliferations without granulomatous inflammation or intralesional nematodes were also found throughout the liver and lungs, suggesting metastasis from the intestine. Immunohistochemical staining of the hepatic and pulmonary tumor tissue with vimentin and S100 suggests a neurofibroblastic origin.

The innate immune response in fetal lung mesenchymal cells targets VEGFR2 expression and activity.

In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Vegfr2 Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme.

The Stromal Intervention: Regulation of Immunity and Inflammation at the Epithelial-Mesenchymal Barrier.

The immune system safeguards organ integrity by employing a balancing act of inflammatory and immunosuppressive mechanisms designed to neutralize foreign invaders and resolve injury. Maintaining or restoring a state of immune homeostasis is particularly challenging at barrier sites where constant exposure to immunogenic environmental agents may induce destructive inflammation. Recent studies underscore the role of epithelial and mesenchymal barrier cells in regulating immune cell function and local homeostatic and inflammatory responses. Here, we highlight immunoregulatory circuits engaging epithelial and mesenchymal cells in the intestine, airways, and skin and discuss how immune communications with hematopoietic cells and the microbiota orchestrate local immune homeostasis and inflammation.

Immunoexpression of tumour necrosis factor-α, interleukin-1α and interleukin-10 on odontogenic cysts and tumours.

AIM: To analyse the immunoreactivity of IL-1α, TNF-α and IL-10 in odontogenic cysts and tumours and to investigate possible associations with established biological behaviours of these different lesions. METHODOLOGY: Immunohistochemical expression of anti-IL-1α, anti-TNF-α and anti-IL-10 antibodies was assessed on epithelium and mesenchyme of 20 radicular cysts (RCs), 20 residual cysts (RECs), 20 dentigerous cysts (DCs), 18 solid ameloblastomas (SAs), 20 keratocystic odontogenic tumours (KCOTs) and 15 dental follicles (DFs). Comparative analysis of data was performed using the nonparametric Wilcoxon signed-rank test and Kruskal-Wallis's test. RESULTS: Significantly greater expression of IL-1α in the epithelium was noted in RC, KCOT and SA (P = 0.01), whilst IL-10 and TNF-α was in the epithelium of RC, DC and KCOT (P < 0.01). In the mesenchyme, significantly greater immunopositivity was observed for IL-1α, IL-10 and TNF-α in KCOT, DC and RC (P < 0.01). In epithelial and mesenchymal tissues, there were a significant number of cases of RC and DC with IL-1α < IL-10 ratio (P < 0.01), whilst SA and KCOT showed IL-1α > IL-10 (P < 0.01). There was a significantly greater percentage of DF, DC and KCOT with TNF-α > IL10 ratio (P < 0.01). CONCLUSION: These results suggest involvement of the proteins in the pathogenesis of odontogenic cysts and tumours, with emphasis on the highest immunoreactivity of osteolysis stimulating factors in tumours with aggressive biological behaviour, such as SA and KCOT.

Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells.

Follicular dendritic cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat. Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of marginal reticular cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. Rather, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population.

Epithelial-mesenchymal transition induces an antitumor immune response mediated by NKG2D receptor.

Epithelial-mesenchymal transition (EMT) is a morphogenetic process characterized by the acquisition of mesenchymal properties linked with an invasive phenotype and metastasis of tumor cells. NK group 2, member D (NKG2D) is an NK cell-activating receptor crucially involved in cancer immunosurveillance. In this study, we show that induction of EMT by TGF-ß stimulation of human keratinocytes, by glycogen synthase kinase-3ß inhibition in several epithelial tumor cell lines, and by Snail1 overexpression in colorectal cancer cells strongly upregulated the expression of NKG2D ligands (NKG2DLs), MHC class I chain-related molecules A and B (MICA/B) and ULBP1-3. Overexpression of Snail1 and inhibition of glycogen synthase kinase-3ß in colorectal tumor cells markedly induced the activity of Sp1 transcription factor, which plays a key role in the upregulation of NKG2DL expression during EMT. The stimulation of MICA/B expression by TGF-ß treatment was independent of Sp1, but it involved posttranslational mechanisms mediated by mammalian target of rapamycin pathway. Accordingly, with the increased expression of NKG2DLs, triggering of EMT rendered cancer cells more susceptible to NKG2D-mediated killing by NK cells. In agreement, MICA/B were expressed in vivo in well-differentiated colorectal tumors with retained epithelial characteristics, whereas no expression of MICA/B was detected in poorly differentiated and invasive colorectal tumors that have lost epithelial characteristics. This decrease of MICA/B expression was associated with a dramatic increase of NKG2D(+)-tumor infiltrating lymphocytes. Overall, our findings indicate that EMT is a relevant checkpoint in the control of tumor progression through NKG2D-mediated immune responses.

Mesenchymal contribution to recruitment, infiltration, and positioning of leukocytes in human melanoma tissues.

To understand factors that regulate leukocyte entry and positioning within human melanoma tissues, we performed a multiparametric quantitative analysis of two separated regions: the intratumoral area and the peritumoral stroma. Using two mesenchymal markers, fibroblast activation protein (FAP) and CD90, we identified three subsets of mesenchymal cells (MCs): (i) intratumoral FAP(+)CD90(low/-) MC, (ii) peritumoral FAP(+)CD90(+) MC, and (iii) FAP(-)CD90(+) perivascular MC. We characterized CD90(+) MCs, which showed a stable CCL2-secretory phenotype when long-term expanded ex vivo, and heavily surrounded peritumoral Duffy antigen receptor for chemokine(+) (DARC) postcapillary venules, supporting a role for these vessels in peritumoral inflammatory leukocyte recruitment. Conversely, the intratumoral area was variably invaded by FAP(+)CD90(low/-) MCs that colocalized with a distinct extracellular matrix (ECM) network. A positive correlation was observed between intratumoral stromal cell/ECM networks and leukocyte infiltration among tumor cells (TCs), as well as in a stroma-dependent xenograft tumor model. Adoptively transferred T lymphocytes preferentially infiltrated tumors composed of TC+MC, compared with TCs only. Altogether, our results suggest that a variety of MCs contribute to regulate different steps of leukocyte tumor infiltration, that is, CD90(+) cells surrounding peritumoral vessels secrete CCL2 to recruit CCR2(+) leukocytes at the tumor periphery, whereas intratumoral FAP(+) cells organize a stromal scaffold that contact guide further invasion among densely packed tumor cells.

Immune heterogeneity of glioblastoma subtypes: extrapolation from the cancer genome atlas.

PURPOSE: The molecular heterogeneity of glioblastoma has been well recognized and has resulted in the generation of molecularly defined subtypes. These subtypes (classical, neural, mesenchymal, and proneural) are associated with particular signaling pathways and differential patient survival. Less understood is the correlation between these glioblastoma subtypes with immune system effector responses, immune suppression and tumor-associated and tumor-specific antigens. The role of the immune system is becoming increasingly relevant to treatment as new agents are being developed to target mediators of tumor-induced immune suppression which is well documented in glioblastoma. EXPERIMENTAL DESIGN: To ascertain the association of antigen expression, immune suppression, and effector response genes within glioblastoma subtypes, we analyzed the Cancer Genome Atlas (TCGA) glioblastoma database. RESULTS: We found an enrichment of genes within the mesenchymal subtype that are reflective of anti-tumor proinflammatory responses, including both adaptive and innate immunity and immune suppression. CONCLUSIONS: These results indicate that distinct glioma antigens and immune genes demonstrate differential expression between glioblastoma subtypes and this may influence responses to immune therapeutic strategies in patients depending on the subtype of glioblastoma they harbor.

Potently immunosuppressive 5-fluorouracil-resistant mesenchymal stromal cells completely remit an experimental autoimmune disease.

We treated mice with 5-fluorouracil (5-FU) to isolate a quiescent and undifferentiated mesenchymal stromal cell (MSC) population from the bone marrow. We examined these 5-FU-resistant MSCs (5-FU-MSCs) free from hematopoietic components for CFU fibroblasts (CFU-Fs) and assessed their immunosuppressive potential in vitro and in vivo. We differentiated fibroblastic CFU-Fs (Fibro-CFU-Fs) from mixed CFU-Fs, based on the absence of in situ expression of CD11b and CD45 hematopoietic markers, as well as on their differentiation capacity. Fibro-CFU-Fs were associated with increased numbers of large-sized Fibro-CFU-Fs (≥9 mm(2)) that displayed enhanced capacity for differentiation into adipogenic and osteogenic mesenchymal lineages. Administration of these 5-FU-resistant CD11b(-)CD45(-) MSCs 6 d after myelin oligodendrocyte glycoprotein (MOG) immunization completely remitted MOG-induced experimental autoimmune encephalomyelitis after initial development of mild disease. The remission was accompanied by reduced CNS cellular infiltration and demyelination, as well as a significant reduction in anti-MOG Ab and splenocyte proliferation to MOG. MOG-stimulated splenocytes from these mice showed elevated levels of Th2 cytokines (IL-4, IL-5, and IL-6) and decreased IL-17. Compared with untreated MSCs, 5-FU-MSCs demonstrated potent immunosuppression of Con A-stimulated splenocytes in vitro, even at a 1:320 MSC/splenocyte ratio. Immunosuppression was accompanied by elevated IL-1ra, IL-10, and PGE(2). Blocking IL-1ra, IL-10, and PGE(2), but not IL-6, heme oxygenase-1, and NO, attenuated 5-FU-MSC-induced immunosuppression. Together, our findings suggested that immunosuppression by 5-FU-MSC is mediated by a combination of elevated IL-1ra, IL-10, and PGE(2), anti-inflammatory Th2 cytokines, and decreased IL-17. Our findings suggested that 5-FU treatment identifies a population of potently immunosuppressive 5-FU-MSCs that have the potential to be exploited to remit autoimmune diseases.

Human mesenchymal stromal cells express CD14 cross-reactive epitopes.

Mesenchymal stromal cells (MSCs) do not express a unique definite epitope or marker gene. As such, minimal criteria were recently established for defining multipotent MSC. These criteria include expression of CD73, CD90, CD105, and a lack of hematopoietic marker expression. However, we detected binding of a CD14 antibody on bone marrow- and placenta-derived MSC and investigated the staining of CD14 antibodies on these MSC in more detail. The MSC were isolated from human bone marrow and placenta tissue, expanded, characterized by quantitative RT-PCR, flow cytometry, and immunocytochemistry and differentiated to generate osteoblasts, chondrocytes, and adipocytes. The CD14-cross-reactive MSCs were enriched by cell sorting. Human peripheral blood mononuclear cells, fibroblasts, and hematopoietic cell lines served as controls. Utilizing four different clones of CD14 monoclonal antibodies, we found that three CD14 reagents stained the MSC. Two CD14 antibodies (HCD14 and M5E2) clearly marked the CD90(+) MSC population with distinct intensities, clone 134 620 generated a shift in flow cytometry histograms, but clone MΦP9 did not stain MSC. Transcripts encoding CD14 or the CD14 protein were not detected in MSC. We confirm that bone marrow- and placenta-derived MSC do not express CD14 and that the CD14 antibody MΦP9 discriminates between monocytes and MSC more efficiently than the other antibodies employed here. This investigation does not contradict previous work but provides a more accurate characterization of MSC.

Mesenchymal cells of the intestinal lamina propria.

The mesenchymal elements of the intestinal lamina propria reviewed here are the myofibroblasts, fibroblasts, mural cells (pericytes) of the vasculature, bone marrow-derived stromal stem cells, smooth muscle of the muscularis mucosae, and smooth muscle surrounding the lymphatic lacteals. These cells share similar marker molecules, origins, and coordinated biological functions previously ascribed solely to subepithelial myofibroblasts. We review the functional anatomy of intestinal mesenchymal cells and describe what is known about their origin in the embryo and their replacement in adults. As part of their putative role in intestinal mucosal morphogenesis, we consider the intestinal stem cell niche. Lastly, we review emerging information about myofibroblasts as nonprofessional immune cells that may be important as an alarm system for the gut and as a participant in peripheral immune tolerance.

Latent infection by γherpesvirus stimulates profibrotic mediator release from multiple cell types.

Although γherpesvirus infections are associated with enhanced lung fibrosis in both clinical and animal studies, there is limited understanding about fibrotic effects of γherpesviruses on cell types present in the lung, particularly during latent infection. Wild-type mice were intranasally infected with a murine γherpesvirus (γHV-68) or mock-infected with saline. Twenty-eight days postinfection (dpi), ∼14 days following clearance of the lytic infection, alveolar macrophages (AMs), mesenchymal cells, and CD19-enriched cell populations from the lung and spleen express M(3) and/or glycoprotein B (gB) viral mRNA and harbor viral genome. AMs from infected mice express more transforming growth factor (TGF)-ß(1), CCL2, CCL12, TNF-α, and IFN-γ than AMs from mock-infected mice. Mesenchymal cells express more total TGF-ß(1), CCL12, and TNF-α than mesenchymal cells from mock-infected mice. Lung and spleen CD19-enriched cells express more total TGF-ß(1) 28 dpi compared with controls. The CD19-negative fraction of the spleen overexpresses TGF-ß(1) and harbors viral genome, but this likely represents infection of monocytes. Purified T cells from the lung harbor almost no viral genome. Purified T cells overexpress IL-10 but not TGF-ß(1). Intracellular cytokine staining demonstrated that lung T cells at 28 dpi produce IFN-γ but not IL-4. Thus infection with a murine γherpesvirus is sufficient to upregulate profibrotic and proinflammatory factors in a variety of lung resident and circulating cell types 28 dpi. Our results provide new information about possible contributions of these cells to fibrogenesis in the lungs of individuals harboring a γherpesvirus infection and may help explain why γHV-68 infection can augment or exacerbate fibrotic responses in mice.

Immunotherapy in the context of hematopoietic stem cell transplantation: the emerging role of natural killer cells and mesenchymal stromal cells.

Immunotherapy in the context of hematopoietic stem cell transplantation has been dominated for many years by T-cell- and dendritic-cell-based treatment modalities. During the last decade, insight into the biology of natural killer (NK) cells and mesenchymal stromal cells (MSC) has rapidly increased and resulted in NK- and MSC-based therapeutic strategies in clinical practice. This article reviews current knowledge of the biology and clinical aspects of NK cells and MSC.

Cellular mechanisms of TNF function in models of inflammation and autoimmunity.

The TNF/TNF receptor (TNFR) system has a prominent role in the pathogenesis of chronic inflammatory and autoimmune disorders. Extensive research in animal models with deregulated TNF expression has documented that TNF may initiate or sustain inflammatory pathology, while at the same time may exert immunomodulatory or disease-suppressive activities. The TNF/TNFR system encompassing both the soluble and the transmembrane form of TNF with differential biological activities, as well as the differential usage of its receptors, mediating distinct functions, appears to confer complexity but also specificity in the action of TNF. The inherent complexity in TNF-mediated pathophysiology highlights the requirement to address the role of TNF taking into account both proinflammatory tissue-damaging and immunomodulatory functions in a cellular and receptor-specific manner. In this review, we discuss our current understanding of the involvement of TNF in chronic inflammation and autoimmunity, focusing on TNF-mediated cellular pathways leading to the pathogenesis or progression of joint and intestinal inflammatory pathology. Knowledge of the mechanisms by which TNF either initiates or contributes to disease pathology is fundamentally required for the design of safe and effective anti-TNF/TNFR therapies for human inflammatory and autoimmune disorders.

RCAS1, MT, and vimentin as potential markers of tumor microenvironment remodeling.

A tumor stimulates the remodeling of its microenvironment for its own survival. To protect its own growth and induce angiogenesis, the tumor changes the structure of extracellular matrix and the function of existing cells; it thus chemo-attracts immune system cells altering their function. In our study, we discuss the potential markers of tumor microenvironment remodeling. For instance, RCAS1 is a protein responsible for tumor escape from host immunologic surveillance that additionally seems to be involved in the remodeling of the microenvironment. Another protein, metallothionein, which is both anti-apoptotic and pro-proliferative, is also responsible for modulating the response of immune system cells. Most likely, the expression of this protein by the fibroblasts of tumor microenvironment is related to the remodeled phenotype of these cells because of the tumor influence on cancer-associated fibroblasts. Lastly, vimentin is a protein that would appear to be the marker for the mesenchymal transition of cells from the epithelial phenotype. These cells seem to acquire the mesenchymal phenotype to migrate so that they can facilitate the development of metastases. Interestingly, the expression of vimentin has also been observed in the tumor microenvironment as well and may serve as a marker of a remodeled stroma in the process of facilitating tumor spread.

TGF-beta1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1beta but not abrogated by corticosteroids.

BACKGROUND: Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFbeta1 stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1beta. METHODS: BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFbeta1 and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1beta. Results were analyzed using non-parametric statistical tests. RESULTS: Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFbeta1 significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFbeta1 then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and alpha-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFbeta1 induced transition but IL-1beta enhanced the transition. CONCLUSION: Our results indicate, that TGFbeta1 can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFbeta1 in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.

Mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from several human tissues and expanded ex vivo for clinical use. MSCs are identified by their adherent properties, immunephenotype, and differentiation potential. MSCs display immunological properties that have been demonstrated both in vitro and in vivo, in animal models and in humans, although the exact mechanisms underlying these effects remain largely unknown. MSCs preferentially home to damaged tissue and secrete paracrine factors with anti-inflammatory properties. The immunomodulatory and reparative and anti-inflammatory properties of MSCs have been tested in a variety of animal models and have been applied in specific clinical settings. Potential clinical applications of MSCs include prevention and treatment of therapy-resistant acute graft-versus-host disease, prevention and treatment of rejection after either hematopoieitc stem cell and solid organ transplantation, tissue repair, and treatment of inborn errors and autoimmune diseases. This review focuses on recent advances that have broadened our understanding of the biological and functional properties of MSCs, which are increasingly attracting the attention of researchers involved in the optimization of approaches for reparative and regenerative cell therapy, as well as in the perspective of modulating immune response against alloantigens or, even, autoantigens.