Samia Q. Khan

Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1.

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a−/−) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFβ signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a−/− animals. Treatment of podocytes in vitro with TGF-β1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-β1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-&bgr;, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

A Small Molecule β2 Integrin Agonist Improves Chronic Kidney Allograft Survival by Reducing Leukocyte Recruitment and Accompanying Vasculopathy.

Kidney allograft rejection is associated with infiltration of inflammatory CD11b+ leukocytes. A CD11b agonist leukadherin-1 (LA1) increases leukocyte adhesion, preventing their transmigration and tissue recruitment in vivo. Here, we test the extent to which LA1-mediated activation of CD11b/CD18 enhances kidney allograft survival in a mouse model of fully MHC-mismatched orthotopic kidney transplantation, where C57BL/6J (H-2b) recipients received kidney allografts from Balb/c mice (H-2d). Isograft control recipients received a kidney from a littermate. Control isograft and allograft recipients were treated daily with cyclosporine (CsA) for 2 weeks, while the test group received CsA therapy and daily LA1 injections during week 1 and alternate days during weeks 2–8. LA1 treatment reduced interstitial leukocyte infiltration in the allograft, reduced neointimal hyperplasia and glomerular damage, and prolonged graft survival from 48.5% (CsA only) to 100% (CsA and LA1) on day 60. Serum creatinine levels showed significantly improved kidney function in LA1-treated mice compared to CsA-treated allograft controls [0.52 ± 0.18 mg/dL vs 0.24 ± 0.07 mg/dL (n = 5), respectively]. Furthermore, combination therapy reduced macrophage infiltration and increased the frequency of FoxP3 + Tregs in the allograft. These findings indicate a crucial role for CD11b/CD18 in the control of leukocyte migration to the transplanted kidney and identify integrin agonist LA1 as a novel potential therapeutic agent for kidney transplantation.

Response to Taraban, Ferdinand, and Al-Shamkhani

We demonstrated that the TNF receptor superfamily member 25 (TNFRSF25) agonistic antibody 4C12 drives the in vivo proliferation of preexisting CD4+FoxP3+ cells (Tregs) but not that of conventional CD4 cells (Tconvs) (1). TNFRSF25-mediated Treg proliferation depends upon MHC II loaded with self-antigen and is blocked by calcineurin inhibitors. If antigen is provided to Tconvs in vitro or in vivo, TNFRSF25 signals also costimulate proliferation and effector cell activity (2–5). Thus, Treg proliferation to TNFRSF25 signals is due to the continuous presence of self-antigenic stimulation of the TCR in vivo, rather than to the level of TNFRSF25 expression. TNFRSF25-mediated Treg expansion is not unique to the agonistic 4C12 antibody, as a TL1A-Ig fusion protein mediates similar Treg expansion in vivo (unpublished observations). In support of this conclusion, the subsequent reports by Al-Shamkhani’s group and Siegel’s group demonstrated that in transgenic mouse models overexpressing TL1A, Tregs proliferate at high frequencies throughout the secondary lymphoid organs and have increased frequencies of Tconvs with an effector/memory phenotype systemically, resulting in mice that are prone to spontaneous inflammatory bowel disease (6, 7). Meylan et al. also demonstrate that TL1A inhibits the de novo induction of Tregs from Tconvs in vitro (7). Both of these recent reports agree that TNFRSF25 attenuates the suppressive capacity of Tregs. In a letter to the JCI, Al-Shamkhani and colleagues provide evidence that the level of expression of TNFRSF25 appears much more similar between Tregs and Tconvs than we originally reported. In recent studies, we have further explored the binding of additional anti-TNFRSF25 antibodies and TL1A-Ig fusion proteins generated in our laboratory to Tregs and Tconvs by flow cytometry. We have found clone-specific variations in the intensity of TNFRSF25 staining between Tconvs and Tregs, which suggests that, as indicated by Al-Shamkhani, the absolute expression of TNFRSF25 between Tconvs and Tregs is not a key determinant in the proliferative specificity of TNFRSF25 agonists or TL1A to Tregs. Instead, as both our and Siegel’s group concluded, the determining factor of TNFRSF25 action in vivo is the availability of cognate antigen, with self-antigen continuously present for Treg expansion. Together, these three reports agree that TNFRSF25 is a regulator of Treg proliferative responses, both with the natural ligand (TL1A) and with receptor agonistic reagents (antibodies and fusion proteins). In the transgenic systems used by Taraban and colleagues and Meylan et al., the continuous availability of TL1A stimulates both the proliferation of Tregs and activation of antigen-experienced effector/memory phenotype Tconvs systemically (6, 7). The finding by Meylan et al. with regard to inhibition of in vitro iTreg generation by TL1A is consistent with the possibility of a role for TL1A in inflammatory bowel disease, in which tolerance to endogenous microbial antigens may have been disrupted by transgenic overexpression of TL1A (7). The balance of TNFRSF25 triggering regulatory or effector immunity is likely dependent upon the availability of cognate antigen to Tregs or Tconvs and not on absolute differences in the expression of the receptor between each subset.

Abstract B03: Integrin agonists reduce infiltration of tumor-associated macrophages to promote T-cell mediated anti-tumor immunity

Tumors associated macrophages (TAMs) mediate neovascularization and promote tumor growth. Integrin CD11b/CD18 is highly expressed on cells of myeloid and granulocytic lineage and mediates their adhesion, migration and tissue recruitment functions. CD11b+ TAMs are present at high numbers in several types of human cancers, including breast and non-small cell lung cancer, and are associated with reduced T-cell mediated anti-tumor immunity. This suggests that reducing infiltration of CD11b+ TAMs could have a therapeutic benefit. Recently, we have developed a novel small molecule CD11b agonist termed leukadherin-1 (LA1) that binds to and allosterically activates integrin CD11b on leukocytes. We also reported that CD11b activation is a more effective strategy for reducing CD11b+ cell tissue infiltration, as compared to integrin antagonists. LA1 binding transiently increases leukocyte adhesion to the vascular endothelium and reduces their transmigration in vivo. Here, we show that LA1 mediated CD11b activation reduced the number of CD11b+ TAMs in two different murine tumor models that was accompanied with diminished primary tumor growth. LA1 treated animals also had significantly reduced tumor angiogenesis and prolonged survival. These improved aspects were accompanied with reduction in vessel density as indicated by reduced intratumoral expression levels of CD31 and αSMA and increased numbers of CD8+ T cells indicating an anti-tumor immune profile response fostering tumor suppression. Our data provide a rationale for using allosteric CD11b agonists to target TAMs as a novel therapeutic approach for treating cancer. Citation Format: Samia Q. Khan, Mohd. H. Faridi, Shehryar J. Khaliqdina, Antonio J. Barbosa, Judith A. Varner, Vineet Gupta. Integrin agonists reduce infiltration of tumor-associated macrophages to promote T-cell mediated anti-tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B03.

Abstract A17: An integrin agonist suppresses breast cancer by reducing CD11b+ leukocytes, promoting anti-tumor immunity

Breast cancer remains the second highest cancer-related cause of death in women today. Neoadjuvant therapies reduce tumor size in patients but long-term survival remains poor. Tumor microenvironment shows a significant infiltration of CD11b+ tumor associated macrophages (TAMs) that promote tumor growth and metastases, while suppressing T-cell mediated anti-tumor immunity, suggesting that reducing infiltration of CD11b+ TAMs could have a therapeutic benefit. We recently developed a novel small molecule that targets CD11b+ cells and reduces their migration and tissue recruitment. Unlike previously described strategies, our methodology involves allosteric activation of CD11b, which induces transient increase in adhesion of CD11b+ cells to the vascular endothelium, thereby reducing the ability of the myeloid cells to transmigrate out of circulation and suppressing their recruitment into tumor and inflamed tissues. However, it is currently not known as to whether small molecule mediated CD11b activation can reduce TAM infiltration in tumors and whether that will have a therapeutic benefit. Here, we present results from studies with our lead compound leukadherin1 (LA1) in murine models of breast cancer. Our studies show that treatment of established syngeneic Cl66 orthotopic breast tumors in Balb/c mice with LA1 significantly reduces tumor growth and increases survival. LA1 treatment significantly reduced infiltration of CD11b+ cells into the tumor stroma, without affecting the levels of cells in circulation. LA1 treatment also significantly reduced expression of molecules that promote angiogenesis and tumor growth including MMP9, S100A8 and ARG1 that coincided with decreased CD31 and α-SMA, suggesting tumor vasculature disruption. Immune cell profiling revealed that LA1 treatment further promotes anti-tumor immunity by increasing the influx of cytotoxic CD8+ T cells into tumors. These data provide a rationale for using allosteric CD11b agonists to target TAM recruitment as a novel therapeutic approach for breast cancer. Citation Format: Samia Q. Khan, Mohd H. Faridi, Brian Ruffell, Ana M. S. Lopez, Shehryar Jehangir, Antonio J. Barbosa, Marta Torroella-Kouri, Lisa M. Coussens, Judith Varner, Vineet Gupta. An integrin agonist suppresses breast cancer by reducing CD11b+ leukocytes, promoting anti-tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A17.