Cutting Edge: CXCR3 Escapes X Chromosome Inactivation in T Cells during Infection: Potential Implications for Sex Differences in Immune Responses.

CXCR3, an X-linked gene, is subject to X chromosome inactivation (XCI), but it is unclear whether CXCR3 escapes XCI in immune cells. We determined whether CXCR3 escapes XCI in vivo, evaluated the contribution of allelic CXCR3 expression to the phenotypic properties of T cells during experimental infection with Leishmania, and examined the potential implications to sex differences in immune responses. We used a bicistronic CXCR3 dual-reporter mouse, with each CXCR3 allele linked to a green or red fluorescent reporter without affecting endogenous CXCR3 expression. Our results show that CXCR3 escapes XCI, biallelic CXCR3-expressing T cells produce more CXCR3 protein than monoallelic CXCR3-expressing cells, and biallelic CXCR3-expressing T cells produce more IFN-γ, IL-2, and CD69 compared with T cells that express CXCR3 from one allele during Leishmania mexicana infection. These results demonstrate that XCI escape by CXCR3 potentially contributes to the sex-associated bias observed during infection.

Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung.

The complement system plays a critical role in immune responses against pathogens. However, its identity and regulation in the lung are not fully understood. This study aimed to explore the role of tripartite motif protein (TRIM) 72 in regulating complement receptor (CR) of the Ig superfamily (CRIg) in alveolar macrophage (AM) and innate immunity of the lung. Imaging, absorbance quantification, and flow cytometry were used to evaluate in vitro and in vivo AM phagocytosis with normal, or altered, TRIM72 expression. Pulldown, coimmunoprecipitation, and gradient binding assays were applied to examine TRIM72 and CRIg interaction. A pneumonia model was established by intratracheal injection of Pseudomonas aeruginosa. Mortality, lung bacterial burden, and cytokine levels in BAL fluid and lung tissues were examined. Our data show that TRIM72 inhibited CR-mediated phagocytosis, and release of TRIM72 inhibition led to increased AM phagocytosis. Biochemical assays identified CRIg as a binding partner of TRIM72, and TRIM72 inhibited formation of the CRIg-phagosome. Genetic ablation of TRIM72 led to improved pathogen clearance, reduced cytokine storm, and improved survival in murine models of severe pneumonia, specificity of which was confirmed by adoptive transfer of wild-type or TRIM72KO AMs to AM-depleted TRIM72KO mice. TRIM72 overexpression promoted bacteria-induced NF-κB activation in murine alveolar macrophage cells. Our data revealed a quiescent, noninflammatory bacterial clearance mechanism in the lung via AM CRIg, which is suppressed by TRIM72. In vivo data suggest that targeted suppression of TRIM72 in AM may be an effective measure to treat fatal pulmonary bacterial infections.

IL-17A promotes susceptibility during experimental visceral leishmaniasis caused by Leishmania donovani.

Leishmania donovani is an intracellular parasite that infects professional phagocytes and causes visceral leishmaniasis (VL). The immune response during VL has been extensively studied in the context of T-helper (Th)1 and Th2 responses. Immunity against this parasite is dependent on IFN-γ production and subsequent macrophage activation, and the Th2 response promotes granuloma formation. The cytokine IL-17A is associated with neutrophilic inflammation. Depletion of neutrophils during experimental VL results in enhanced parasitic loads. Furthermore, although patients resistant to VL showed enhanced levels of IL-17A in circulation, little is known about the role of IL-17A during VL infection. Here, we used IL-17A-deficient mice and IL-17A reporter mice to address the role of IL-17A during VL. IL-17A(-/-) mice were highly resistant to VL infection, showing decreased parasites in the liver and spleen. This unexpected phenotype was associated with enhanced IFN-γ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in reduced number of granulomas. We also found γδ T and Th17 cells as the main IL-17A(+) cells during VL infection. Our data reveal an unexpected role of IL-17A rendering susceptibility against L. donovani by regulating the IFN-γ response and promoting detrimental inflammation.

Pentalinonsterol, a Constituent of Pentalinon andrieuxii, Possesses Potent Immunomodulatory Activity and Primes T Cell Immune Responses.

The use of natural products as adjuvants has emerged as a promising approach for the development of effective vaccine formulations. Pentalinonsterol (PEN) is a recently isolated compound from the roots of Pentalinon andrieuxii and has been shown to possess antileishmanial activity against Leishmania spp. The objective of this study was to examine the immunomodulatory properties of PEN and evaluate its potential as an adjuvant. Macrophages and bone-marrow-derived dendritic cells (BMDCs) were stimulated with PEN and tested for gene expression, cytokine production, and their ability to activate T cells in vitro. PEN was also evaluated for its ability to generate antigen-specific Th1 and Th2 responses in vivo, following ovalbumin (OVA) immunization using PEN as an adjuvant. The results obtained demonstrate that PEN enhances the expression of NF-κB and AP1 transcription factors, promotes gene expression of Tnfα, Il6, Nos2, and Arg1, and upregulates MHCII, CD80, and CD86 in macrophages. PEN also enhanced IL-12 production in BMDCs and promoted BMDC-mediated production of IFN-γ by T cells. Further, mice immunized with OVA and PEN showed enhanced antigen-specific Th1 and Th2 cytokines in their splenocytes and lymph node cells, as well as increased levels of IgG1 and IgG2 in their sera. Taken together, this study demonstrates that PEN is a potent immunomodulatory compound and potentially can be used as an adjuvant for vaccine development against infectious diseases.

Deletion of macrophage migration inhibitory factor inhibits murine oral carcinogenesis: Potential role for chronic pro-inflammatory immune mediators.

Oral cancer kills about 1 person every hour each day in the United States and is the sixth most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1ß, Tnf-α, chemokines Cxcl1, Cxcl6 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, systemic accumulation of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression of chronic pro-inflammatory immune mediators. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer.

CXCR3 expression defines a novel subset of innate CD8+ T cells that enhance immunity against bacterial infection and cancer upon stimulation with IL-15.

Innate CD8(+) T cells are a heterogeneous population with developmental pathways distinct from conventional CD8(+) T cells. However, their biology, classification, and functions remain incompletely understood. We recently demonstrated the existence of a novel population of chemokine (C-X-C motif) receptor 3 (CXCR3)-positive innate CD8(+) T cells. Here, we investigated the functional properties of this subset and identified effector molecules and pathways which mediate their function. Adoptive transfer of IL-15 activated CXCR3(+) innate CD8(+) T cells conferred increased protection against Listeria monocytogenes infection in susceptible IFN-γ(-/-) mice compared with similarly activated CXCR3(-) subset. This was associated with enhanced proliferation and IFN-γ production in CXCR3(+) cells. Further, CXCR3(+) innate cells showed enhanced cytotoxicity against a tumor cell line in vitro. In depth analysis of the CXCR3(+) subset showed increased gene expression of Ccl5, Klrc1, CtsW, GP49a, IL-2Rß, Atp5e, and Ly6c but reduced IFN-γR2 and Art2b. Ingenuity pathway analysis revealed an up-regulation of genes associated with T-cell activation, proliferation, cytotoxicity, and translational initiation in CXCR3(+) populations. Our results demonstrate that CXCR3 expression in innate CD8(+) T cells defines a subset with enhanced cytotoxic potential and protective antibacterial immune functions. Immunotherapeutic approaches against infectious disease and cancer could utilize CXCR3(+) innate CD8(+) T-cell populations as novel clinical intervention strategies.

Characterization of cross-protection by genetically modified live-attenuated Leishmania donovani parasites against Leishmania mexicana.

Previously, we showed that genetically modified live-attenuated Leishmania donovani parasite cell lines (LdCen(-/-) and Ldp27(-/-)) induce a strong cellular immunity and provide protection against visceral leishmaniasis in mice. In this study, we explored the mechanism of cross-protection against cutaneous lesion-causing Leishmania mexicana. Upon challenge with wild-type L. mexicana, mice immunized either for short or long periods showed significant protection. Immunohistochemical analysis of ears from immunized/challenged mice exhibited significant influx of macrophages, as well as cells expressing MHC class II and inducible NO synthase, suggesting an induction of potent host-protective proinflammatory responses. In contrast, substantial inhibition of IL-10, IL-4, and IL-13 expression and the absence of degranulated mast cells and less influx of eosinophils within the ears of immunized/challenged mice suggested a controlled anti-inflammatory response. L. mexicana Ag-stimulated lymph node cell culture from the immunized/challenged mice revealed induction of IFN-γ secretion by the CD4 and CD8 T cells compared with non-immunized/challenged mice. We also observed suppression of Th2 cytokines in the culture supernatants of immunized/challenged lymph nodes compared with non-immunized/challenged mice. Adoptively transferred total T cells from immunized mice conferred strong protection in recipient mice against L. mexicana infection, suggesting that attenuated L. donovani can provide protection against heterologous L. mexicana parasites by induction of a strong T cell response. Furthermore, bone marrow-derived dendritic cells infected with LdCen(-/-) and Ldp27(-/-) parasites were capable of inducing a strong proinflammatory response leading to the proliferation of Th1 cells. These studies demonstrate the potential of live-attenuated L. donovani parasites as pan-Leishmania species vaccines.

Transgenic expression of CXCR3 on T cells enhances susceptibility to cutaneous Leishmania major infection by inhibiting monocyte maturation and promoting a Th2 response.

Cutaneous leishmaniasis, caused mainly by Leishmania major, an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major. Furthermore, T cells from L. major-susceptible BALB/c but not L. major-resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3(Tg)) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major-infected BALB/c-CXCR3(Tg) mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3(Tg) mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major.

Signal transducer and activator of transcription 1 (STAT-1) plays a critical role in control of Trypanosoma cruzi infection.

The control of Trypanosoma cruzi infection is related to interferon-γ (IFN-γ) activation leading to intracellular clearance of parasites. The transcription factor signal transducer and activator of transcription 1 (STAT-1) is a key mediator of IFN-γ intracellular signalling and knockout of this protein leads to susceptibility to several intracellular microbes. To determine the role of STAT-1 in host susceptibility to T. cruzi infection we compared the survival, parasite loads and balance of IFN-γ and interleukin-10 (IL-10) responses between wild-type and STAT-1 knockout mice. We found that the lack of STAT-1 resulted in a more robust infection, leading to higher levels of blood and tissue parasites and markedly reduced survival. In addition, infected STAT-1 knockout mice had higher systemic levels of both IFN-γ and IL-10, suggesting that the absence of STAT-1 leads to a disequilibrium of pro-inflammatory and anti-inflammatory cytokines. Analysis of spleen cells indicates that CD4, CD8 cells generate IFN-γ and natural killer cells express IL-13 in STAT-1 knockout animals. The production of IL-17 is particularly enhanced in the absence STAT-1 expression but did not reduce mortality. Overall these results indicate that STAT-1 is important for the control of T. cruzi infection in mice.

STAT4 is critical for immunity but not for antileishmanial activity of antimonials in experimental visceral leishmaniasis.

We and others have previously shown that IL-12 is indispensable for immunity and is required for the optimal antiparasitic activity of antimonials in experimental visceral leishmaniasis caused by Leishmania donovani. Here we investigated the role of STAT4 in immunity against L. donovani using STAT4 knockout mice and also determined the effect of STAT4 deficiency in response to antimonial therapy. Upon infection with L. donovani, stat4⁻/⁻ BALB/c and C57BL/6 mice showed enhanced susceptibility to Leishmania during late time points of infection which was associated with a marked reduction in Th1 responses and hepatic immunopathology. Interestingly, these defects in Th1 responses in stat4⁻/⁻ did not impair the antimonial chemotherapy as both stat4⁻/⁻ and WT mice showed comparable levels of parasite clearance from the liver and spleen. These findings highlight the role of STAT4 in immunity to L. donovani infection and also provide evidence that STAT4 is dispensable for antimonial-based chemotherapy.

Distinct populations of innate CD8+ T cells revealed in a CXCR3 reporter mouse.

CXCR3, expressed mainly on activated T and NK cells, is implicated in a host of immunological conditions and can contribute either to disease resolution or pathology. We report the generation and characterization of a novel CXCR3 internal ribosome entry site bicistronic enhanced GFP reporter (CIBER) mouse in which enhanced GFP expression correlates with surface levels of CXCR3. Using CIBER mice, we identified two distinct populations of innate CD8(+) T cells based on constitutive expression of CXCR3. We demonstrate that CXCR3(+) innate CD8(+) T cells preferentially express higher levels of Ly6C and CD122, but lower levels of CCR9 compared with CXCR3(-) innate CD8(+) T cells. Furthermore, we show that CXCR3(+) innate CD8(+) T cells express higher transcript levels of antiapoptotic but lower levels of proapoptotic factors, respond more robustly to IL-2 and IL-15, and produce significantly more IFN-γ and granzyme B. Interestingly, CXCR3(+) innate CD8(+) T cells do not respond to IL-12 or IL-18 alone, but produce significant amounts of IFN-γ on stimulation with a combination of these cytokines. Taken together, these findings demonstrate that CXCR3(+) and CXCR3(-) innate CD8(+) T cells are phenotypically and functionally distinct. These newly generated CIBER mice provide a novel tool for studying the role of CXCR3 and CXCR3-expressing cells in vivo.

Immunomodulatory and Antileishmanial Activity of Phenylpropanoid Dimers Isolated from Nectandra leucantha.

Three phenylpropanoid dimers (1-3) including two new metabolites were isolated from the extract of the twigs of Nectandra leucantha using antileishmanial bioassay-guided fractionation. The in vitro antiparasitic activity of the isolated compounds against Leishmania donovani parasites and mammalian cytotoxicity and immunomodulatory effects were evaluated. Compounds 1-3 were effective against the intracellular amastigotes within macrophages, with IC50 values of 26.7, 17.8, and 101.9 µM, respectively. The mammalian cytotoxicity, given by the 50% cytotoxic concentration (CC50), was evaluated against peritoneal macrophages. Compounds 1 and 3 were not toxic up to 290 µM, whereas compound 2 demonstrated a CC50 value of 111.2 µM. Compounds 1-3 also suppressed production of disease exacerbatory cytokines IL-6 and IL-10 but had minimal effect on nitric oxide production in L. donovani-infected macrophages, indicating that antileishmanial activity of these compounds is mediated via an NO-independent mechanism. Therefore, these new natural products could represent promising scaffolds for drug design studies for leishmaniasis.

CXCR3 deficiency enhances tumor progression by promoting macrophage M2 polarization in a murine breast cancer model.

Tumor associated macrophages play a vital role in determining the outcome of breast cancer. We investigated the contribution of the chemokine receptor CXCR3 to antitumor immune responses using a cxcr3 deficient mouse orthotopically injected with a PyMT breast cancer cell line. We observed that cxcr3 deficient mice displayed increased IL-4 production and M2 polarization in the tumors and spleens compared to WT mice injected with PyMT cells. This was accompanied by larger tumor development in cxcr3(-/-) than in WT mice. Further, tumor-promoting myeloid derived immune cell populations accumulated in higher proportions in the spleens of cxcr3 deficient mice. Interestingly, cxcr3(-/-) macrophages displayed a deficiency in up-regulating inducible nitric oxide synthase after stimulation by either IFN-γ or PyMT supernatants. Stimulation of bone marrow derived macrophages by PyMT supernatants also resulted in greater induction of arginase-1 in cxcr3(-/-) than WT mice. Further, cxcr3(-/-) T cells activated with CD3/CD28 in vitro produced greater amounts of IL-4 and IL-10 than T cells from WT mice. Our data suggests that a greater predisposition of cxcr3 deficient macrophages towards M2 polarization contributes to an enhanced tumor promoting environment in cxcr3 deficient mice. Although CXCR3 is known to be expressed on some macrophages, this is the first report that demonstrates a role for CXCR3 in macrophage polarization and subsequent breast tumor outcomes. Targeting CXCR3 could be a potential therapeutic approach in the management of breast cancer tumors.

Helminth-excreted/secreted products are recognized by multiple receptors on DCs to block the TLR response and bias Th2 polarization in a cRAF dependent pathway.

Dendritic cells (DCs) recognize pathogens and initiate the T-cell response. The DC-helminth interaction induces an immature phenotype in DCs; as a result, these DCs display impaired responses to TLR stimulation and prime Th2-type responses. However, the DC receptors and intracellular pathways targeted by helminth molecules and their importance in the initiation of the Th2 response are poorly understood. In this report, we found that products excreted/secreted by Taenia crassiceps (TcES) triggered cRAF phosphorylation through MGL, MR, and TLR2. TcES interfered with the LPS-induced NFκB p65 and p38 MAPK signaling pathways. In addition, TcES-induced cRAF signaling pathway was critical for down-regulation of the TLR-mediated DC maturation and secretion of IL-12 and TNF-α. Finally, we show for the first time that blocking cRAF in DCs abolishes their ability to induce Th2 polarization in vitro after TcES exposure. Our data demonstrate a new mechanism by which helminths target intracellular pathways to block DC maturation and efficiently program Th2 polarization.

Pentalinon andrieuxii root extract is effective in the topical treatment of cutaneous leishmaniasis caused by Leishmania mexicana.

Cutaneous leishmaniasis (CL) manifests as localized skin lesions, which lead to significant tissue destruction and disfigurement. In the Yucatan Peninsula, Mayan traditional healers use Pentalinon andrieuxii Muell.-Arg. (Apocynaceae) roots for the topical treatment of CL. Here, we studied the effect of P. andrieuxii root hexane extract (PARE) on the parasites and host cells in vitro and examined its efficacy in the topical treatment of CL caused by Leishmania mexicana. PARE exhibited potent antiparasitic activity in vitro against promastigotes as well as amastigotes residing in macrophages. Electron microscopy of PARE-treated parasites revealed direct membrane damage. PARE also activated nuclear factor kappaB and enhanced interferon-γ receptor and MHC class II expression and TNF-α production in macrophages. In addition, PARE induced production of the Th1 promoting cytokine IL-12 in dendritic cells as well as enhanced expression of the co-stimulatory molecules CD40, CD80, and CD86. In vivo studies showed that L. mexicana-infected mice treated by topical application of PARE resulted in the significant reduction in lesion size and parasite burden compared to controls. These findings indicate that PARE could be used as an alternative therapy for the topical treatment of CL.

Consecutive low doses of cyclosporine A induce pro-inflammatory cytokines and accelerate allograft skin rejection.

Cyclosporine A (CsA) is a fungus-derived molecule with potent immunosuppressive activity that has been largely used to downregulate cell-mediated immune responses during transplantation. However, previous data have indicated that CsA shows immunomodulatory activity that relays on the antigen concentration and the dose of CsA used. To test the hypothesis that minimal doses of CsA may show different outcomes on grafts, we used an experimental model for skin transplants in mice. ICR outbred mice received skin allografts and were either treated daily with different doses of CsA or left untreated. Untreated mice showed allograft rejection within 14 days, with graft necrosis, infiltration of neutrophils and macrophages and displayed high percentages of CD8+ T cells in the spleens, which were associated with high serum levels of IL-12, IFN-g and TNF-α. As expected, mice treated with therapeutic doses of CsA (15 mg/kg) did not show allograft rejection within the follow-up period of 30 days and displayed the lowest levels of IL-12, IFN-g and TNF-α as well as a reduction in CD8+ lymphocytes. In contrast, mice treated with consecutive minimal doses of CsA (5×10(-55) mg/kg) displayed an acute graft rejection as early as one to five days after skin allograft; they also displayed necrosis and strong inflammatory infiltration that was associated with high levels of IL-12, IFN-g and TNF-α. Moreover, the CD4+ CD25hiFoxP3+ subpopulation of cells in the spleens of these mice was significantly inhibited compared with animals that received the therapeutic treatment of CsA and those treated with placebo. Our data suggest that consecutive, minimal doses of CsA may affect Treg cells and may stimulate innate immunity.

Modulation of dendritic cell responses by parasites: a common strategy to survive.

Parasitic infections are one of the most important causes of morbidity and mortality in our planet and the immune responses triggered by these organisms are critical to determine their outcome. Dendritic cells are key elements for the development of immunity against parasites; they control the responses required to eliminate these pathogens while maintaining host homeostasis. However, there is evidence showing that parasites can influence and regulate dendritic cell function in order to promote a more permissive environment for their survival. In this review we will focus on the strategies protozoan and helminth parasites have developed to interfere with dendritic cell activities as well as in the possible mechanisms involved.

Toxoplasma gondii: impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection.

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF-/- mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF-/- mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.

Taenia crassiceps-Excreted/Secreted Products Induce a Defined MicroRNA Profile that Modulates Inflammatory Properties of Macrophages.

Helminth parasites modulate immune responses in their host to prevent their elimination and to establish chronic infections. Our previous studies indicate that Taenia crassiceps-excreted/secreted antigens (TcES) downregulate inflammatory responses in rodent models of autoimmune diseases, by promoting the generation of alternatively activated-like macrophages (M2) in vivo. However, the molecular mechanisms triggered by TcES that modulate macrophage polarization and inflammatory response remain unclear. Here, we found that, while TcES reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages (BMDM). However, TcES alone or in combination with LPS or IL-4 failed to increase the production of the canonical M1 or M2 markers in BMDM. To further define the anti-inflammatory effect of TcES in the response of LPS-stimulated macrophages, we performed transcriptomic array analyses of mRNA and microRNA to evaluate their levels. Although the addition of TcES to LPS-stimulated BMDM induced modest changes in the inflammatory mRNA profile, it induced the production of mRNAs associated with the activation of different receptors, phagocytosis, and M2-like phenotype. Moreover, we found that TcES induced upregulation of specific microRNAs, including miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced BMDM. These results suggest that TcES can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms and hence reduce detrimental outcomes in hosts running with inflammatory diseases.

MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages.

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/ß complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.