Dying cells program their expedient disposal: serum amyloid P component upregulation in vivo and in vitro induced by photodynamic therapy of cancer.

Serum amyloid P component (SAP) is known as a prototypic acute phase reactant in the mouse and the protein that binds to dying cells securing their swift disposal by phagocytes. Treatment of solid tumors by photodynamic therapy (PDT) triggers SAP production in the liver of host mice, its release in the circulation and accumulation in PDT-targeted lesions. In the present study, mouse Lewis lung carcinoma (LLC) cells treated in vitro by PDT are shown to upregulate their gene encoding SAP. This effect was manifested following PDT treatment mediated by various types of photosensitizers (Photofrin, BPD, mTHPC, ALA). Generated SAP protein was not detected in tissue supernatants but remained localized to producing PDT-treated cells. The upregulation of SAP gene was observed also in untreated IC-21 macrophages after they were co-incubated for 4 h with PDT-treated LLC cells. Based on these findings, SAP that accumulates in PDT-treated tumors may originate from both systemic sources (released from the liver as acute phase reactant) and local sources; the latter could include tumor cells directly sustaining PDT injury and macrophages invading the tumor that become stimulated by signals from these affected tumor cells. Since SAP gene upregulation in LLC cells increased with the lethality of PDT dose used for their treatment, we propose that cells sensing they are inflicted with mortal injury can turn on molecular programs insuring not only that they die an innocuous form of death (apoptosis) but also that once they are dead their elimination is (facilitated by SAP) swift and efficient.     

IL-10 contributes to the inhibition of contact hypersensitivity in mice treated with photodynamic therapy

We have explored the effect of photodynamic therapy (PDT) with verteporfin on the induction and expression of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB) in normal mice and IL-10-deficient mice. Our results indicate that DNFB sensitized mice given PDT with verteporfin and whole body red light irradiation exhibited a significant reduction in CHS compared with control animals. Administration of rIL-12 reversed the effect(s) of PDT as did treatment of mice with anti-IL-10-neutralizing Ab. Knockout mice deficient in IL-10 were found to be resistant to the inhibitory effects of PDT. In vitro proliferative responses using spleen cells from DNFB-sensitized and PDT-treated mice showed a significantly lower response to DNBS as compared with cells from DNFB-sensitized mice or DNFB and PDT-treated IL-10-deficient mice. Finally, naive mice exposed to PDT exhibited an increase in skin IL-10 levels, which peaked between 72 and 120 h post-PDT. Together these data support the role of IL-10 as a key modulator in the inhibition of the CHS response by whole body PDT.     

Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) Induces Exposure and Release of Damage-Associated Molecular Patterns (DAMPs) in Human HeLa Cells

The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.     

Photodynamic-therapy Activates Immune Response by disrupting Immunity Homeostasis of Tumor Cells,which Generates Vaccine for Cancer Therapy

Photodynamic therapy (PDT), a regulatory approved cancer treatment, is reported to be capable of causing immunogenic apoptosis. The current data reveal PDT can cause the dysregulation of “eat me” and “don''t eat me” signal by generating reactive oxygen species (ROS) -mediated endoplasmic reticulum (ER) stress. This dysregulation probably contribute to the increased uptake of PDT-killed Lewis lung carcinoma (LLC) cells by homologous dendritic cells (DCs), accompanied by phenotypic maturation (CD80^high, CD86^high, and CD40^high) and functional stimulation (NO^high, IL-10^absent) of dendritic cells as well as subsequent T-cell responses. Morevover, C57BL/6 mice vaccinated with dendritic cells (DCs) pulsed with PDT-treated LLCs (PDT-DCs) or PDT-treated LLCs alone (PDT-LLCs) exhibited potent immunity against LLC tumors. In the current study, the PDT-induced immune response was characterized as a process related with the dysregulation of “eat me” signal and “don''t eat me” signal, revealing the possibility for developing PDT into an antitumor vaccination strategy for personalized cancer immunotherapy.     

Many faces of DAMPs in cancer therapy

A new concept of immunogenic cell death (ICD) has recently been proposed. The immunogenic characteristics of this cell death mode are mediated mainly by molecules called ‘damage-associated molecular patterns'' (DAMPs), most of which are recognized by pattern recognition receptors. Some DAMPs are actively emitted by cells undergoing ICD (e.g. calreticulin (CRT) and adenosine triphosphate (ATP)), whereas others are emitted passively (e.g. high-mobility group box 1 protein (HMGB1)). Recent studies have demonstrated that these DAMPs play a beneficial role in anti-cancer therapy by interacting with the immune system. The molecular pathways involved in translocation of CRT to the cell surface and secretion of ATP from tumor cells undergoing ICD are being elucidated. However, it has also been shown that the same DAMPs could contribute to progression of cancer and promote resistance to anticancer treatments. In this review, we will critically evaluate the beneficial and detrimental roles of DAMPs in cancer therapy, focusing mainly on CRT, ATP and HMGB1.     

Photodynamic therapy-generated vaccine for cancer therapy

A target tumor-derived whole cancer cell therapeutic vaccine was developed based on an in vitro pre-treatment by photodynamic therapy (PDT) and was investigated using a poorly immunogenic tumor model. The vaccine was produced by incubating in vitro expanded mouse squamous cell carcinoma SCCVII cells for 1 h with photosensitizer benzoporphyrin derivative (BPD), then exposing to light (690 nm, 1 J/cm²) and finally to a lethal X-ray dose. Treatment of established subcutaneous SCCVII tumors growing in syngeneic C3H/HeN mice with 2x10⁷ PDT-vaccine cells per mouse by a peritumoral injection produced a significant therapeutic effect, including growth retardation, regression and cures. Tumor specificity of this PDT-generated vaccine was demonstrated by its ineffectiveness when prepared from a mismatched tumor cell line. Vaccine cells retrieved from the treatment site at 1 h postinjection were intermixed with dendritic cells (DC), exhibited heat shock protein 70 on their surface, and were opsonized by complement C3. Tumor-draining lymph nodes treated by the PDT-vaccine contained dramatically increased numbers of DC as well as B and T lymphocytes (with enlarged memory phenotype fraction in the latter), while high levels of surface-bound C3 were detectable on DC and to a lesser extent on B cells. The PDT-vaccine produced no therapeutic benefit against tumors growing in C3-deficient hosts. It is suggested that surface expression of heat shock proteins and complement opsonization are the two unique features of PDT-treated cells securing avid immune recognition of vaccinated tumor and the development of a strong and effective antitumor adaptive immune response.     

Adjuvant treatment for complement activation increases the effectiveness of photodynamic therapy of solid tumors.

Phototoxic lesions generated in tumor tissue by photodynamic therapy (PDT) are recognized by the host as a threat to the integrity and homeostasis at the affected site. Among the canonical pathways invoked by the host for dealing with this type of challenge is the activation of the complement system, integrating proteins that serve as molecular sensors of danger signals produced by PDT and those initiating signalling cascades coupled into the network of inflammatory and immune responses. Since the activated complement system is a salient participant of the antitumor response produced by PDT, it is worth exploring whether its manipulation can be exploited for the therapeutic benefit. Using mouse tumor models, the present study examined the potential of representative complement-activating agents to act as effective adjuvants to PDT. Tumor-localized treatment with zymosan, an alternative complement pathway activator, reduced the recurrence-rate of PDT-treated tumors, markedly increasing the percentage of permanent cures. In contrast, a similar treatment with heat aggregated gamma globulin (complement activator via the classical pathway) was of no significant benefit as a PDT adjuvant. Systemic complement activation with streptokinase treatment had no detectable effect on complement deposition at the tumor site without PDT, but it augmented the extent of complement activity in PDT-treated tumors. This finding based on immunohistochemistry analysis explains the results of tumor therapy experiments, which showed that systemic treatment with streptokinase or a similar agent, urokinase, enhances the PDT-mediated tumor response. Zymosan and streptokinase administrations produced no beneficial results with PDT of tumors growing in complement-deficient mice. This study, therefore, establishes the potential of complement-activating agents to serve as effective adjuvants to PDT for cancer treatment.     

Specific microtubule-depolymerizing agents augment efficacy of dendritic cell-based cancer vaccines

Background

Damage-associated molecular patterns (DAMPs) are associated with immunogenic cell death and have the ability to enhance maturation and antigen presentation of dendritic cells (DCs). Specific microtubule-depolymerizing agents (MDAs) such as colchicine have been shown to confer anti-cancer activity and also trigger activation of DCs.

Methods

In this study, we evaluated the ability of three MDAs (colchicine and two 2-phenyl-4-quinolone analogues) to induce immunogenic cell death in test tumor cells, activate DCs, and augment T-cell proliferation activity. These MDAs were further evaluated for use as an adjuvant in a tumor cell lysate-pulsed DC vaccine.

Results

The three test phytochemicals considerably increased the expression of DAMPs including HSP70, HSP90 and HMGB1, but had no effect on expression of calreticulin (CRT). DC vaccines pulsed with MDA-treated tumor cell lysates had a significant effect on tumor growth, showed cytotoxic T-lymphocyte activity against tumors, and increased the survival rate of test mice. In vivo antibody depletion experiments suggested that CD8⁺ and NK cells, but not CD4⁺ cells, were the main effector cells responsible for the observed anti-tumor activity. In addition, culture of DCs with GM-CSF and IL-4 during the pulsing and stimulation period significantly increased the production of IL-12 and decreased production of IL-10. MDAs also induced phenotypic maturation of DCs and augmented CD4⁺ and CD8⁺ T-cell proliferation when co-cultured with DCs.

Conclusions

Specific MDAs including the clinical drug, colchicine, can induce immunogenic cell death in tumor cells, and DCs pulsed with MDA-treated tumor cell lysates (TCLs) can generate potent anti-tumor immunity in mice. This approach may warrant future clinical evaluation as a cancer vaccine.     

Functional role of a long non-coding RNA LIFR-AS1/miR-29a/TNFAIP3 axis in colorectal cancer resistance to pohotodynamic therapy

Colorectal Cancer (CRC) is one of the most common digestive system malignant tumors. Recently, PDT has been used as a first-line treatment for colon cancer; however, limited curative effect was obtained due to resistance of CRC to PDT. During the past decades, accumulating CRC-related long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs have been reported to exert diverse functions through various biological processes; their dysregulation might trigger and/or promote the pathological changes. Herein, we performed microarrays analysis to identify dysregulated lncRNAs, miRNAs and mRNAs in PDT-treated HCT116 cells to figure out the lncRNA-miRNA interactions related to the resistance of CRC to PDT treatment, and the downstream mRNA target, as well as the molecular mechanism. We found a total of 1096 lncRNAs dysregulated in PDT-treated CRC HCT116 cells; among them, LIFR-AS1 negatively interacted with miR-29a, one of the dysregulated miRNAs in PDT-treated CRC cells, to affect the resistance of CRC to PDT. LIFR-AS1 knockdown attenuated, whereas miR-29a inhibition enhanced the cellular effect of PDT on HCT116 cell proliferation and apoptosis. Furthermore, among the dysregulated mRNAs, TNFAIP3 was confirmed to be a direct target of miR-29a and exerted a similar effect to LIFR-AS1 on the cellular effects of PDT. In summary, LIFR-AS1 serves as a competitive endogenous RNA (ceRNA) for miR-29a to inhibit its expression and up-regulate downstream target TNFAIP3 expression, finally modulating the resistance of CRC to PDT. We provide an experimental basis for this lncRNA/miRNA/mRNA network being a promising target in CRC resistance to PDT treatment.     

Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.     

Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin

Surface-exposed HSP70 and calreticulin are damage-associated molecular patterns (DAMPs) crucially involved in modulating the success of cancer therapy. Photodynamic therapy (PDT) involves the administration of a photosensitising (PTS) agent followed by visible light-irradiation. The reactive oxygen species that are thus generated directly kill tumours by damaging their microvasculature and inducing a local inflammatory reaction. PDT with the PTS photofrin is associated with DAMPs exposure, but the same is not true for other PTSs. Here, we show that when cancer cells are treated with hypericin-based PDT (Hyp-PDT), they surface-expose both HSP70 and calreticulin (CRT). Induction of CRT exposure was not accompanied by co-exposure of ERp57, but this did not compromise the ability of the exposed CRT to regulate the phagocytosis of Hyp-PDT-treated cancer cells by dendritic cells. Interestingly, we found that Hyp-PDT-induced CRT exposure (in contrast to anthracycline-induced CRT exposure) was independent of the presence of ERp57. Our results indicate that Hyp-PDT is a potential anti-cancer immunogenic modality.     

Induction of arthritis by high mobility group box chromosomal protein 1 is independent of tumour necrosis factor signalling

Introduction

TNFα and high mobility group box chromosomal protein 1 (HMGB1) are two potent proinflammatory cytokines implicated as important mediators of arthritis. Increased levels of these cytokines are found in the joints of rheumatoid arthritis patients, and the cytokines trigger arthritis when applied into the joints of naïve mice. HMGB1 is actively released from immune cells in response to TNFα; once released, HMGB1 in turn induces production of several proinflammatory cytokines – including IL-6 and TNFα – by macrophages. Whether HMGB1-induced arthritis is mediated via the TNFα pathway, however, is unknown. The purpose of the present study was to investigate whether the arthritis-inducing effect of HMGB1 is dependent on TNFα expression in vivo and to assess whether TNFα deficiency affects a proinflammatory cytokine response to HMGB1 in vitro.

Methods

TNFα knockout mice and backcrossed control animals on a C57Bl6 background were injected intraarticularly with 5 μg HMGB1. Joints were dissected 3 days after intraarticular injection and were evaluated histologically by scoring the frequency and severity of arthritis. For in vitro studies, mouse spleen cultures from TNFα knockout mice and from control mice were incubated with different doses of HMGB1, and cell culture supernatants were collected at different time points for analysis of IL-6.

Results

Intraarticular injection of HMGB1 into healthy mouse joints resulted in an overall frequency of 32% to 39% arthritic animals. No significant differences were found with respect to the severity and incidence of synovitis between mice deficient for TNFα (seven out of 18 mice with arthritis) in comparison with control TNFα^+/+ animals (six out of 19). No significant differences were detected between spleen cells from TNFα^+/+ mice versus TNFα^-/- mice regarding IL-6 production upon stimulation with highly purified HMGB1 after 24 hours and 48 hours. Upon stimulation with a suboptimal dose of recombinant HMGB1, however, the splenocytes from TNFα^+/+ animals released significantly more IL-6 than cells from the knockout mice (602 ± 112 pg/ml and 304 ± 50 pg/ml, respectively; P < 0.05).

Conclusion

Our data show that HMGB1-triggered joint inflammation is not mediated via the TNF pathway. Combined with our previous study, we suggest that HMGB1-triggered arthritis is probably mediated through IL-1 activation.     

HMGB1 contributes to the development of acute lung injury after hemorrhage

High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.     

Photodynamic therapy–generated cancer vaccine elicits acute phase and hormonal response in treated mice

Photodynamic therapy (PDT)-generated cancer vaccines have shown promising results in preclinical studies and are being introduced in the clinics. Using an SCCVII mouse squamous cell carcinoma-based whole-cell autologous PDT vaccine model developed in our previous work, we have examined systemic effects in vaccinated mice that could be related to the induction of acute phase response. The upregulation of gene encoding serum amyloid P component (prototypic mouse acute phase reactant) was detected in the liver and to a lesser degree in the tumor of vaccinated mice at 24 h post-PDT vaccine treatment. A strong upregulation of gene for heat shock protein 70 was found in both the liver and tumor of mice at 4 h after their PDT vaccine treatment. Changes in the expression of genes for glucocorticoid-induced leucine zipper and serum- and glucocorticoid-regulated kinase 1 that are highly responsive to glucocorticoid modulation were uncovered in both the tumor and liver of vaccinated mice. A rise in the levels of serum corticosterone was detected in mice at 24 h after PDT vaccine treatment. The results indicate that a sudden appearance of a large number of PDT vaccine cells elicits host responses for securing their optimized clearance, which in addition to producing seminal acute phase reactants includes the engagement of glucocorticoid hormones. It is becoming increasingly clear that a consummate execution of this process of PDT vaccine cell removal is critical for tumor antigen recognition and the attainment of potent antitumor immune response.     

Photodynamic therapy of tumors can lead to development of systemic antigen-specific immune response

Background

The mechanism by which the immune system can effectively recognize and destroy tumors is dependent on recognition of tumor antigens. The molecular identity of a number of these antigens has recently been identified and several immunotherapies have explored them as targets. Photodynamic therapy (PDT) is an anti-cancer modality that uses a non-toxic photosensitizer and visible light to produce cytotoxic reactive oxygen species that destroy tumors. PDT has been shown to lead to local destruction of tumors as well as to induction of anti-tumor immune response.

Methodology/Principal Findings

We used a pair of equally lethal BALB/c colon adenocarcinomas, CT26 wild-type (CT26WT) and CT26.CL25 that expressed a tumor antigen, β-galactosidase (β-gal), and we treated them with vascular PDT. All mice bearing antigen-positive, but not antigen-negative tumors were cured and resistant to rechallenge. T lymphocytes isolated from cured mice were able to specifically lyse antigen positive cells and recognize the epitope derived from beta-galactosidase antigen. PDT was capable of destroying distant, untreated, established, antigen-expressing tumors in 70% of the mice. The remaining 30% escaped destruction due to loss of expression of tumor antigen. The PDT anti-tumor effects were completely abrogated in the absence of the adaptive immune response.

Conclusion

Understanding the role of antigen-expression in PDT immune response may allow application of PDT in metastatic as well as localized disease. To the best of our knowledge, this is the first time that PDT has been shown to lead to systemic, antigen- specific anti-tumor immunity.     

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80(high), CD83(high), CD86(high), MHC-II(high)) and functional stimulation (NO(high), IL-10(absent), IL-1β(high)) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress.     

Characterisation of the Xenogeneic Immune Response to Microencapsulated Fetal Pig Islet-Like Cell Clusters Transplanted into Immunocompetent C57BL/6 Mice

Xenotransplantation of microencapsulated fetal pig islet-like cell clusters (FP ICCs) offers a potential cellular therapy for type 1 diabetes. Although microcapsules prevent direct contact of the host immune system with the xenografted tissue, poor graft survival is still an issue. This study aimed to characterise the nature of the host immune cells present on the engrafted microcapsules and effects on encapsulated FP ICCs that were transplanted into immunocompetent mice. Encapsulated FP ICCs were transplanted into the peritoneal cavity of C57BL/6 mice. Grafts retrieved at days 1, 3, 7, 14 and 21 post-transplantation were analysed for pericapsular fibrotic overgrowth (PFO), cell viability, intragraft porcine gene expression, macrophages, myofibroblasts and intraperitoneal murine cytokines. Graft function was assessed ex vivo by insulin secretion studies. Xenogeneic immune response to encapsulated FP ICCs was associated with enhanced intragraft mRNA expression of porcine antigens MIP-1α, IL-8, HMGB1 and HSP90 seen within the first two weeks post-transplantation. This was associated with the recruitment of host macrophages, infiltration of myofibroblasts and collagen deposition leading to PFO which was evident from day 7 post-transplantation. This was accompanied by a decrease in cell viability and loss of FP ICC architecture. The only pro-inflammatory cytokine detected in the murine peritoneal flushing was TNF-α with levels peaking at day 7 post transplantation. This correlated with the onset of PFO at day 7 implying activated macrophages as its source. The anti-inflammatory cytokines detected were IL-5 and IL-4 with levels peaking at days 1 and 7, respectively. Porcine C-peptide was undetectable at all time points post-transplantation. PFO was absent and murine intraperitoneal cytokines were undetectable when empty microcapsules were transplanted. In conclusion, this study demonstrated that the macrophages are direct effectors of the xenogeneic immune response to encapsulated FP ICCs leading to PFO mediated by a combination of both pro- and anti-inflammatory cytokines.