Macrophage-mediated extracellular matrix remodeling controls host Staphylococcus aureus susceptibility in the skin.

Hypodermis is the predominant site of Staphylococcus aureus infections that cause cellulitis. Given the importance of macrophages in tissue remodeling, we examined the hypodermal macrophages (HDMs) and their impact on host susceptibility to infection. Bulk and single-cell transcriptomics uncovered HDM subsets with CCR2-dichotomy. HDM homeostasis required the fibroblast-derived growth factor CSF1, ablation of which abrogated HDMs from the hypodermal adventitia. Loss of CCR2- HDMs resulted in accumulation of the extracellular matrix component, hyaluronic acid (HA). HDM-mediated HA clearance required sensing by the HA receptor, LYVE-1. Cell-autonomous IGF1 was required for accessibility of AP-1 transcription factor motifs that controlled LYVE-1 expression. Remarkably, loss of HDMs or IGF1 limited Staphylococcus aureus expansion via HA and conferred protection against cellulitis. Our findings reveal a function for macrophages in the regulation of HA with an impact on infection outcomes, which may be harnessed to limit the establishment of infection in the hypodermal niche.

Disruption of the endopeptidase ADAM10-Notch signaling axis leads to skin dysbiosis and innate lymphoid cell-mediated hair follicle destruction.

Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting ß-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.

Low dose radiation primed iNOS + M1macrophages modulate angiogenic programming of tumor derived endothelium.

Solid tumors are covered by stroma, which is hypoxic in nature and composed of various non-malignant components such as endothelial cells, fibroblasts, and pericytes that support tumor growth. Tumor stroma represents a mechanical barrier for tumor infiltration of CD8+ effector T cells in particular. In this context, our previous studies have demonstrated the therapeutic impact of Low-Dose Radiation (LDR)-primed and M1-retuned (iNOS+) peritumoral macrophages that produce inducible nitric oxide, have immunological roles on tumor infiltration of effector T cells, cancer-related inflammation, and subsequent tumor immune rejection in a mouse model of pancreatic cancer. These findings suggested a possible modification of tumor endothelium by LDR-primed macrophages. In line with these observations, here we demonstrate the influence of LDR in down-modulating HIF-1 in irradiated tumors in the course of polarization of irradiated tumor-associated macrophages toward an M1 phenotype. Furthermore, we demonstrate that M1 macrophages which are primed by LDR can directly influence angiogenic responses in eNOS+ endothelial cells which produce nitric oxide having both vascular and physiological roles. Furthermore, we demonstrate that naïve macrophages, upon differentiating to an M1 phenotype either by Th1 stimuli or LDR, potentially modify sphingosine-1-phosphate/VEGF-induced angiogenic signaling in tumor-derived endothelial cells with tumorigenic potential, thus indicating the significance of iNOS+ macrophages in modulating signaling in eNOS+ tumor-derived endothelium. Our study suggests that iNOS+ macrophages can activate tumor endothelium which may contribute to cancer-directed immunotherapy in particular.

Low doses of gamma irradiation potentially modifies immunosuppressive tumor microenvironment by retuning tumor-associated macrophages: lesson from insulinoma.

Tumor infiltrating iNOS+ macrophages under the influence of immunosuppressive tumor microenvironment gets polarized to tumor-promoting and immunosuppressive macrophages, known as tumor-associated macrophages (TAM). Their recruitment and increased density in the plethora of tumors has been associated with poor prognosis in cancer patients. Therefore, retuning of TAM to M1 phenotype would be a key for effective immunotherapy. Radiotherapy has been a potential non-invasive strategy to improve cancer immunotherapy and tumor immune rejection. Irradiation of late-stage tumor-bearing Rip1-Tag5 mice twice with 2 Gy dose resulted in profound changes in the inflammatory tumor micromilieu, characterized by induction of M1-associated effecter cytokines as well as reduction in protumorigenic and M2-associated effecter cytokines. Similarly, in vitro irradiation of macrophages with 2 Gy dose-induced expression of iNOS, NO, NFκBpp65, pSTAT3 and proinflammatory cytokines secretion while downregulating p38MAPK which are involved in iNOS translation and acquisition of an M1-like phenotype. Enhancement of various M2 effecter cytokines and angiogenic reprogramming in iNOs+ macrophage depleted tumors and their subsequent reduction by 2 Gy dose in Rip1-Tag5 transgenic mice furthermore demonstrated a critical role of peritumoral macrophages in the course of gamma irradiation mediated M1 retuning of insulinoma.

Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells.

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and ß1 and ß3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.

Development of a rapid in vitro pre-screen for distinguishing effective liposome-adjuvant delivery systems.

Liposomes are a strong supporting tool in vaccine technology, as they are a versatile system that not only act as antigen delivery systems but also adjuvants that can be highly effective at stimulating both innate and adaptive immune responses. Their ability to induce cell-mediated immunity makes their use in vaccines a useful tool in the development of novel, more effective vaccines against intracellular infections (e.g. HIV, malaria and tuberculosis). Currently, screening of novel liposome formulations uses murine in vivo models which generate data that often correlates poorly with human data. In addition, these models are both high cost and low throughput, making them prohibitive for large scale screening of formulation libraries. This study uses the cationic liposome formulation DDA:TDB (known as cationic adjuvant formulation 01 (CAF01)), as a lead formulation, along with other liposome formulations of known in vivo efficacy to develop an in vitro screening tool for liposome formulation development. THP-1-derived macrophages were the model antigen presenting cell used to assess the ability of the liposome formulations to attract, associate with and activate antigen presenting cells in vitro, crucial steps necessary for an effective immune response to antigen. By using a combination of in vitro functions, the study highlights the potential use of an in vitro screening tool, to predict the in vivo efficacy of novel liposome formulations. CAF01 was predicted as the most effective liposome formulation when assessing all in vitro functions and a measure of in vitro activation was able to predict 80% of the liposome correctly for their ability to induce an in vivo IFN-Ò¯ response.

Emerging neo adjuvants for harnessing therapeutic potential of M1 tumor associated macrophages (TAM) against solid tumors: Enusage of plasticity.

Macrophages are a major component of the tumor microenvironment (TME) of most tumors. They are characterized by a high degree of functional plasticity which enable these cells to both promote and eliminate established tumors. Under the influence of immunosuppressive TME, tumor infiltrating iNOS+ and CD11b+ M-1 effector macrophages get polarized towards tumor associated macrophages (TAM) which are tropic to variety of tumors. Increased infiltration and density of TAM is associated with tumor progression and poor prognosis in the plethora of tumors due to their angiogenetic and tissue re-modelling nature. Importantly, TAMs are also responsible for developing endothelium anergy, a major physical barrier for majority of cancer directed immune/chemotherapies. Therefore, functional retuning/re-educating TAM to M-1 phenotypic macrophages is paramount for effective immunotherapy against established tumors. In this review, we discuss and provide comprehensive update on TAM-targeted approaches for enhancing immunity against various solid tumors.

A single aromatic residue in transcriptional repressor protein KorA is critical for cooperativity with its co-regulator KorB.

A central feature of broad host range IncP-1 plasmids is the set of regulatory circuits that tightly control plasmid core functions under steady-state conditions. Cooperativity between KorB and either KorA or TrbA repressor proteins is a key element of these circuits and deletion analysis has implicated the conserved C-terminal domain of KorA and TrbA in this interaction. By NMR we show that KorA and KorB interact directly and identify KorA amino acids that are affected on KorB binding. Studies on mutants showed that tyrosine 84 (or phenylalanine, in some alleles) is dispensable for repressor activity but critical for the specific interaction with KorB in both in vivo reporter gene assays and in vitro electrophoretic mobility shift and co-purification assays. This confirms that direct and specific protein-protein interactions are responsible for the cooperativity observed between KorB and its corepressors and lays the basis for determining the biological importance of this cooperativity.

Sphingosine-1-Phosphate (S-1P) Promotes Differentiation of Naive Macrophages and Enhances Protective Immunity Against Mycobacterium tuberculosis.

Sphingosine-1-phosphate (S-1P) is a key sphingolipid involved in the pathobiology of various respiratory diseases. We have previously demonstrated the significance of S-1P in controlling non-pathogenic mycobacterial infection in macrophages, and here we demonstrate the therapeutic potential of S-1P against pathogenic Mycobacterium tuberculosis (H37Rv) in the mouse model of infection. Our study revealed that S-1P is involved in the expression of iNOS proteins in macrophages, their polarization toward M1 phenotype, and secretion of interferon (IFN)-γ during the course of infection. S-1P is also capable of enhancing infiltration of pulmonary CD11b+ macrophages and expression of S-1P receptor-3 (S-1PR3) in the lungs during the course of infection. We further revealed the influence of S-1P on major signaling components of inflammatory signaling pathways during M. tuberculosis infection, thus highlighting antimycobacterial potential of S-1P in animals. Our data suggest that enhancing S-1P levels by sphingolipid mimetic compounds/drugs can be used as an immunoadjuvant for boosting immunity against pathogenic mycobacteria.

Podophyllotoxin and Rutin Modulate M1 (iNOS+) Macrophages and Mitigate Lethal Radiation (LR) Induced Inflammatory Responses in Mice.

Accidental exposure to lethal doses of Gamma radiation leads to the systemic inflammatory syndrome which causes mortality. In view of this, management of hemopoietic syndrome by modulating pro-inflammatory response in clinically manageable time period seems to be the most appropriate strategy for encountering radiation induced damage and recovery. As both tissue and peripheral macrophages are critical for the management of radiation induced injuries, we have unraveled the immunomodulatory potential of radioprotective formulation (G-003M) on peripheral macrophages populations in this study. G-003M inhibited lethal radiation induced NO and Th1 effector cytokines in the exposed macrophages indicating its M1 dim polarizing capacity. In similar lines, conditioning of mice with G-003M before lethal irradiation (LR) inhibited LR induced titre of Th1 effector cytokines in both serums as well as in lung, small intestine, and spleen tissue confirming its immunomodulatory potential. G-003M potentially down modulated inflammatory response in LPS induced inflammatory model and enhanced M2 polarization of iNOS+ M1 effector macrophages providing a molecular hint on G-003M mechanism of action on macrophages. These observations revealed that G-003M potentially modulate pro-inflammatory programming of macrophages and mitigate radiation-induced inflammatory stress which is believed to contribute significantly to radioprotective attribute of G-003M. In this study, we demonstrate that Rutin and Podophyllotoxin drive M1dim/M2 polarization of LR primed macrophages apart from protecting DNA from radiation. These drugs have the capacity to programme innate immune cells like macrophages which may be involved in homeostasis during recovery.

Poly-N-acryloyl-(l-phenylalanine methyl ester) hollow core nanocapsules facilitate sustained delivery of immunomodulatory drugs and exhibit adjuvant properties.

Polymeric hollow nanocapsules have attracted significant research attention as novel drug carriers and their preparation is of particular concern owing to the feasibility to encapsulate a broad range of drug molecules. This work presents for the first time the synthesis and development of novel poly-N-acryloyl l-phenylalanine methyl ester hollow core nanocapsules (NAPA-HPNs) of avg. size ca. 100-150 nm by the mini-emulsion technique. NAPA-HPNs are biocompatible and capable of encapsulating sodium nitroprusside (SNP) at a rate of ∼1.3 µM per mg of capsules. These NAPA-HPNs + SNP nano-formulations maintained homeostasis of macrophages which carry and facilitate the action of various drug molecules used against various diseases. These NAPA-HPNs also facilitate the prolonged release of a low level of nitric oxide (NO) and enhance the metabolic activities of pro-inflammatory macrophages, which are important for the action of various drugs in body fluids. NAPA-HPN mediated skewing of naïve macrophages toward the M1 phenotype potentially demonstrates its adjuvant action on the innate immune system. These results potentially suggested that NAPA-HPNs can serve both as a carrier of drugs as well as an adjuvant for the immune system. Thus, these nanocapsules could be used for the effective management of various infectious or tumor diseases where immune-stimulation is paramount for treatment.

Inhibitors of Apoptosis Protein Antagonists (Smac Mimetic Compounds) Control Polarization of Macrophages during Microbial Challenge and Sterile Inflammatory Responses.

Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity.

CD14/TLR4 priming potentially recalibrates and exerts anti-tumor efficacy in tumor associated macrophages in a mouse model of pancreatic carcinoma.

Pancreatic cancer is the fourth major cause of cancer related deaths in the world and 5 year survival is below 5%. Among various tumor directed therapies, stimulation of Toll-like receptors (TLR) has shown promising effects in various tumor models. However, pancreatic cancer cells frequently express these receptors themselves and their stimulation (TLR 2 and/or 4 particularly) within tumor microenvironment is known to potentially enhance tumor cell proliferation and cancer progression. Consistent stimulation of tumor associated macrophages (TAMs), in particular with tumor derived TLR ligand within the tumor microenvironment promotes cancer related inflammation, which is sterile, non-immunogenic and carcinogenic in nature. In view of this, recalibrating of TAM has the potential to induce immunogenic inflammation. Consistent with this, we provide experimental evidence for the first time in this study that priming of TAMs with TLR4 ligend (LPS) alone or in combination with IFN-γ not only recalibrates pancreatic tumor cells induced M2 polarization, but also confers anti-tumor potential in TAMs. Most interestingly, reduced tumor growth in macrophage depleted animals suggests that macrophage directed approaches are important for the management of pancreatic tumors.

Designing liposomal adjuvants for the next generation of vaccines.

Liposomes not only offer the ability to enhance drug delivery, but can effectively act as vaccine delivery systems and adjuvants. Their flexibility in size, charge, bilayer rigidity and composition allow for targeted antigen delivery via a range of administration routes. In the development of liposomal adjuvants, the type of immune response promoted has been linked to their physico-chemical characteristics, with the size and charge of the liposomal particles impacting on liposome biodistribution, exposure in the lymph nodes and recruitment of the innate immune system. The addition of immunostimulatory agents can further potentiate their immunogenic properties. Here, we outline the attributes that should be considered in the design and manufacture of liposomal adjuvants for the delivery of sub-unit and nucleic acid based vaccines.