The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.

Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malaria but their role in leishmaniasis is unclear. In this study, we report a detrimental role of Ly6Chi iMOs in visceral leishmaniasis (VL) caused by Leishmania donovani. We demonstrate that Ly6Chi iMOs are continuously recruited into the spleen and liver during L. donovani infection and they are preferential targets for the parasite. Using microarray-based gene expression profiling, we show that Ly6Chi iMOs isolated from the infected liver and spleen have distinct phenotypic and activation profiles. Furthermore, we demonstrate that blocking the recruitment of Ly6Chi iMOs into the liver and spleen during L. donovani infection using a CCR2 antagonist reduces the frequency of the pathogenic IFN-γ/IL10 dual producer CD4+ T cells in the spleen and leads to a significant reduction in parasite loads in the liver and spleen. Using STAT1−/− mice we show that STAT1 is critical for mediating the recruitment of Ly6Chi iMOs into organs during L. donovani infection, and adaptive transfer of wild type Ly6Chi iMOs into STAT1−/− recipients renders them susceptible to disease. Our findings reveal an unexpected pathogenic role for Ly6Chi iMOs in promoting parasite survival in VL and open the possibility of targeting this population for host-directed therapy during VL.

Tumor-induced expansion of Tregs is a significant obstacle to cancer immunotherapy. However, traditional approaches to deplete Tregs are often inefficient, provoking autoimmunity. We show here that administration of IL-27-expressing recombinant adeno-associated virus (AAV-IL-27) significantly inhibits tumor growth and enhances T cell responses in tumors. Strikingly, we found that AAV-IL-27 treatment causes rapid depletion of Tregs in peripheral blood, lymphoid organs, and - most pronouncedly - tumor microenvironment. AAV-IL-27-mediated Treg depletion is dependent on IL-27 receptor and Stat1 in Tregs and is a combined result of CD25 downregulation in Tregs and inhibition of IL-2 production by T cells. In combination with a GM-CSF vaccine, AAV-IL-27 treatment not only induced nearly complete tumor rejection, but also resulted in amplified neoantigen-specific T cell responses. AAV-IL-27 also dramatically increased the efficacy of anti-PD-1 therapy, presumably due to induction of PD-L1 in T cells and depletion of Tregs. Importantly, AAV-IL-27 therapy did not induce significant adverse events, partially due to its induction of IL-10. In a plasmacytoma mouse model, we found that IL-10 was required for AAV-IL-27-mediated tumor rejection. Thus, our study demonstrates the potential of AAV-IL-27 as an independent cancer therapeutic and as an efficient adjuvant for cancer immunotherapy.

Objective. Systemic lupus erythematosus (SLE) develops much more readily in females than in males. Previous research has focused primarily on identifying mechanisms pertinent to the pathology in females. The aim of the current study was to delineate active protective mechanisms in males. We present evidence of a new male-associated mechanism of protection against the development of lupus-like disease in lupus-prone (NZB ؋ NZW)F1 mice.Methods. We identified previously uncharacterized cellular and functional differences in myeloid cells between male and female (NZB ؋ NZW)F1 mice, with the use of flow cytometry, confocal imaging, in vivo antibody-mediated depletion, and in vitro cell coculture assays.Results. A population of Gr-1 high Ly-6G؉CD11b؉ myeloid cells was found to be constitutively increased in male (NZB ؋ NZW)F1 mice as compared with female mice and was regulated by testosterone. The cells were located adjacent to spleen B cell follicles in vivo and were found to directly inhibit cytokine-induced differentiation of naive B cells into antibody-secreting cells in vitro. Most notably, treatment with anti-Gr-1-depleting antibodies increased the spontaneous production of antinuclear autoantibodies in male (NZB ؋ NZW)F1 mice, while a similar approach in female mice had no effect on disease development. Conclusion. Male lupus-prone (NZB ؋ NZW)F1mice harbor elevated levels of a population of myeloid cells with pronounced immunosuppressive capacities that specifically target B cells and the production of antibodies in vivo. We suggest that these cells represent a male-driven inhibitory mechanism involved in the control of B cell pathogenesis, delaying (or preventing) lupus-like disease development in otherwise genetically predisposed male (NZB ؋ NZW)F1 mice.Systemic lupus erythematosus (SLE) is a prototypical systemic autoimmune disorder. The disease is characterized by elevated levels of antinuclear antibodies (ANAs) and cellular infiltration of various organs, including the skin, heart, and kidney (1). Like most systemic autoimmune diseases, SLE predominantly affects women. The female-to-male ratio of newly diagnosed patients reaches 9:1 between the ages of 15 years and 45 years (2,3). This coincides with the years when female sex hormones are the most active, and thus, much research has focused on elucidating the role of female sex hormones (particularly estrogen) in SLE. For example, it is well accepted that elevated levels of estrogens affect B lymphocyte selection processes by altering the threshold for negative selection, thereby allowing more self-reactive B cells to survive and differentiate (4,5). Testosterone, on the other hand, is believed to be protective, based on mouse studies (6-9) and clinical data from SLE patients treated with testosterone or the adrenal steroid dehydroepiandrosterone (10-12). Still, the target of regulation and the specific mechanism of protection mediated by testosterone remain unidentified.In some animal models of SLE, female predominance is similarly observed. One such mode...

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.—Terrazas, C., Varikuti, S., Kimble, J., Moretti, E., Boyaka, P. N., Satoskar, A. R., IL‐17A promotes susceptibility during experimental visceral leishmaniasis caused by Leishmania donovani. FASEB J. 30, 1135‐1143 (2016). http://www.fasebj.org

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.

Background Ibrutinib is a Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible kinase (ITK) inhibitor used for treating chronic lymphocytic leukaemia (CLL) and other cancers. Although ibrutinib is known to inhibit the growth of breast cancer cell growth in vitro, its impact on the treatment and metastasis of breast cancer is unclear. Methods Using an orthotopic mouse breast cancer model, we show that ibrutinib inhibits the progression and metastasis of breast cancer. Results Ibrutinib inhibited proliferation of cancer cells in vitro, and Ibrutinib-treated mice displayed significantly lower tumour burdens and metastasis compared to controls. Furthermore, the spleens and tumours from Ibrutinib-treated mice contained more mature DCs and lower numbers of myeloid-derived suppressor cells (MDSCs), which promote disease progression and are linked to poor prognosis. We also confirmed that ex vivo treatment of MDSCs with ibrutinib switched their phenotype to mature DCs and significantly enhanced MHCII expression. Further, ibrutinib treatment promoted T cell proliferation and effector functions leading to the induction of antitumour TH1 and CTL immune responses. Conclusions Ibrutinib inhibits tumour development and metastasis in breast cancer by promoting the development of mature DCs from MDSCs and hence could be a novel therapeutic agent for the treatment of breast cancer.

Cancers of the oral cavity remain the sixth most diagnosed cancer worldwide, with high rates of recurrence and mortality. We determined the role of STAT1 during oral carcinogenesis using two orthotopic models in mice genetically deficient for Stat1. Metastatic (LY2) and nonmetastatic (B4B8) head and neck squamous cell carcinoma (HNSCC) cell lines were injected into the oral cavity of Stat1 deficient (Stat1−/−) and Stat1 competent (Stat1+/+) mice. Stat1−/− mice displayed increased tumor growth and metastasis compared to Stat1+/+ mice. Mechanistically, Stat1−/− mice displayed impaired CD4+ and CD8+ T‐cell expansion compared to Stat1+/+ mice. This was associated with enhanced T‐cell exhaustion, and severely attenuated T‐cell antitumor effector responses including reduced expression of IFN‐γ and perforin at the tumor site. Interestingly, tumor necrosis factor (TNF)‐α production by T cells in tumor‐bearing mice was suppressed by Stat1 deficiency. This deficiency in T‐cell expansion and functional responses in mice was linked to PD‐1 and CD69 overexpression in T cells of Stat1−/− mice. In contrast, we observed increased accumulation of CD11b+ Ly6G+ myeloid derived suppressor cells in tumors, draining lymph nodes, spleens and bone marrow of tumor‐bearing Stat1−/− mice, resulting in a protumorigenic microenvironment. Our data demonstrates that STAT1 is an essential mediator of the antitumor response through inhibition of myeloid derived suppressor cell accumulation and promotion of T‐cell mediated immune responses in murine head and neck squamous cell carcinoma. Selective induction of STAT1 phosphorylation in HNSCC patients could potentially improve oral tumor outcomes and response to therapy.