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Myeloid cells are recruited to damaged tissues where they can resolve infections and tumor growth or stimulate wound healing and tumor progression. Recruitment of these cells is regulated by integrins, a family of adhesion receptors that includes integrin CD11b. Here we report that, unexpectedly, integrin CD11b does not regulate myeloid cell recruitment to tumors but instead controls myeloid cell polarization and tumor growth. CD11b activation promotes pro-inflammatory macrophage polarization by stimulating expression of microRNA Let7a . In contrast, inhibition of CD11b prevents Let7a expression and induces cMyc expression, leading to immune suppressive macrophage polarization, vascular maturation, and accelerated tumor growth. Pharmacological activation of CD11b with a small molecule agonist, Leukadherin 1 (LA1), promotes pro-inflammatory macrophage polarization and suppresses tumor growth in animal models of murine and human cancer. These studies identify CD11b as negative regulator of immune suppression and a target for cancer immune therapy. Recruitment of myeloid cells can be regulated by integrin CD11b. Here the authors show that in the tumor microenvironment, CD11b is not essential for recruitment of myeloid cells but rather induces macrophage anti-tumorigenic polarization via stimulating let7a and NFκB signaling and that pharmacological activation of CD11b enhances survival in mouse models of cancer.

Background Osteoarthritis (OA) is a painful degenerative joint disease and a leading source of years lived with disability globally due to inadequate treatment options. Neuroimmune interactions reportedly contribute to OA pain pathogenesis. Notably, in rodents, macrophages in the DRG are associated with onset of persistent OA pain. Our objective was to determine the effects of acute systemic macrophage depletion on pain-related behaviors and joint damage using surgical mouse models in both sexes. Methods We depleted CSF1R + macrophages by treating male macrophage Fas-induced apoptosis (MaFIA) transgenic mice 8- or 16-weeks post destabilization of the medial meniscus (DMM) with AP20187 or vehicle control (10 mg/kg i.p. , 1x/day for 5 days), or treating female MaFIA mice 12 weeks post partial meniscectomy (PMX) with AP20187 or vehicle control. We measured pain-related behaviors 1–3 days before and after depletion, and, 3–4 days after the last injection we examined joint histopathology and performed flow cytometry of the dorsal root ganglia (DRGs). In a separate cohort of male 8-week DMM mice or age-matched naïve vehicle controls, we conducted DRG bulk RNA-sequencing analyses after the 5-day vehicle or AP20187 treatment. Results Eight- and 16-weeks post DMM in male mice, AP20187-induced macrophage depletion resulted in attenuated mechanical allodynia and knee hyperalgesia. Female mice showed alleviation of mechanical allodynia, knee hyperalgesia, and weight bearing deficits after macrophage depletion at 12 weeks post PMX. Macrophage depletion did not affect the degree of cartilage degeneration, osteophyte width, or synovitis in either sex. Flow cytometry of the DRG revealed that macrophages and neutrophils were reduced after AP20187 treatment. In addition, in the DRG, only MHCII + M1-like macrophages were significantly decreased, while CD163 + MHCII- M2-like macrophages were not affected in both sexes. DRG bulk RNA-seq revealed that Cxcl10 and Il1b were upregulated with DMM surgery compared to naïve mice, and downregulated in DMM after acute macrophage depletion. Conclusions Acute systemic macrophage depletion reduced the levels of pro-inflammatory macrophages in the DRG and alleviated pain-related behaviors in established surgically induced OA in mice of both sexes, without affecting joint damage. Overall, these studies provide insight into immune cell regulation in the DRG during OA.

Diabetic glomerular injury is a major complication of diabetes mellitus and is the leading cause of end stage renal disease (ESRD). Healthy podocytes are essential for glomerular function and health. Injury or loss of these cells results in increased proteinuria and kidney dysfunction and is a common finding in various glomerulopathies. Thus, mechanistic understanding of pathways that protect podocytes from damage are essential for development of future therapeutics. MicroRNA-146a (miR-146a) is a negative regulator of inflammation and is highly expressed in myeloid cells and podocytes. We previously reported that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). Here we report generation of mice with selective deletion of miR-146a in podocytes and use of these mice in models of glomerular injury. Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a –/– ) animals via administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice, suggesting that podocyte-expressed miR-146a protects these cells, and thus glomeruli, from damage. Furthermore, induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals, further confirming the protective role of podocyte-expressed miR-146a. We also confirmed that the direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli and erlotinib, an ErbB4 and EGFR inhibitor, reducedits upregulation and the proteinuria in treated animals. Primary miR146 –/– podocytes from these animals also showed a basally upregulated TGFβ-Smad3 signaling in vitro . Taken together, this study shows that podocyte-specific miR-146a is imperative for protecting podocytes from glomerular damage, via modulation of ErbB4/EGFR, TGFβ, and linked downstream signaling.

Globally, cancer is among the leading causes of death with 1 in 6 deaths due to cancer. Lung cancer is leading cause of cancer-related death accounting for more deaths than breast, prostate, and colon cancer combined. Current treatment options for lung cancer patients vary by stage and type of disease, but include surgical resection whenever possible, chemotherapy and radiotherapy when applicable. These treatments help control disease progression, but lack specificity and have unwanted side effects. Retraining the immune system to respond to cancer is an attractive alternative to chemo- or radio- therapies because it has the ability to circumvent some of these severe side effects. For example, immune checkpoint inhibitors (e.g., anti-PD1, anti-CTLA-4, etc.), work by augmenting the anti-tumor T cell response, and have had a significant impact on improving the survival of lung cancer patients. Although, these new therapies have done well to improve lung cancer patient survival, there is still more room for development of targeted therapeutics that improve patient outcomes.Beyond T cell targeted immunotherapy, there are increasing efforts to find other immune cells as targets for new immunotherapies. Lung tumors have large numbers of tumor associated macrophages (TAMs) which suppress the adaptive immune response, increase neo-vascularization of the tumor, and secrete pro-tumor factors to promote tumor growth. Integrin CD11b/CD18 (also known as Mac-1, CR3 and αMβ2) is highly expressed on myeloid cells, including TAMs, and plays an important role in innate cell recruitment and other biological functions. GB1275 (previously known as ADH-503) is a novel small molecule CD11b allosteric modulator. GB1275 is currently in Phase 1/2 clinical development for various cancer types. Here, we describe our pre-clinical studies to investigate the impact of CD11b modulation in the context of lung cancer.To assess the role of CD11b in regulating lung tumor growth, we studied lung tumor growth in the absence of CD11b using CD11b knockout (KO) mice, by pharmacologically modulating CD11b in WT mice by treatment with GB1275, or in the context of genetically modulated CD11b using CD11b knock-in (KI) mice. All tumors were propagated in the Lewis lung carcinoma (LLC) syngeneic tumor model. LLC tumors grew faster and had heavier tumor burden in CD11b KO mice as compared to WT controls. In contrast to CD11b KO mice, pharmacologic modulation of CD11b by GB1275 treatment significantly reduced tumor growth. We developed a transgenic CD11b knock-in (KI) mouse that renders CD11b in a partially active state by introducing a point mutation at residue 332 of the ligand binding CD11b A/I-domain, changing isoleucine to glycine (I332G). CD11b KI mice showed a significant reduction in both the size and the rate of LLC tumor growth, as compared to the WT mice, mimicking treatment effects with GB1275. Tumor immune profiling showed a significant reduction in monocyte, granulocyte, and mature TAM infiltration in GB1275-treated and CD11b KI tumors. Dendritic cell populations changed to enhance antigen cross presentation as a result of CD11b modulation. There was an increase in the frequency of CD8+ cytotoxic T lymphocytes and decrease of FoxP3+ regulatory T cells in GB1275-treated and CD11b KI tumors. CD11b modulation affected TAM polarization manifested by increased frequencies of M1-like F4/80+MHCII+ TAMs and decreased M2-like F4/80+CD206+ TAMs and from gene expression profiling of CD11b-modulated macrophages. CD11b-modulated macrophages showed significant downregulation of Ccl2 mRNA levels, which resulted in less CCL2 protein secreted into circulation. Taken together, these results indicate that CD11b modulation not only reprograms TAMs to enhance the T cell response, but also works by reducing trafficking of new TAMs in the lung tumor microenvironment to reduce the overall inflammatory burden.Overall, CD11b modulation affects both monocyte/macrophage recruitment to lung tumors and reprograms TAMs to enhance the anti-tumor immune response. There are many antagonism approaches to inhibit pro-tumoral TAMs, but here we describe an agonistic approach via CD11b modulation. As CD11b+ cells are highly abundant in human lung tumors, targeting CD11b via allosteric modulation is a novel therapeutic strategy against lung cancer.

Osteoarthritis (OA) is a painful degenerative joint disease and a leading source of years lived with disability globally due to inadequate treatment options. Neuroimmune interactions reportedly contribute to OA pain pathogenesis. Notably, in rodents, macrophages in the DRG are associated with onset of persistent OA pain. Our objective was to determine the effects of acute systemic macrophage depletion on pain-related behaviors and joint damage using surgical mouse models in both sexes. We depleted CSF1R+ macrophages by treating male macrophage Fas-induced apoptosis (MaFIA) transgenic mice 8- or 16-weeks destabilization of the medial meniscus (DMM) with AP20187 or vehicle control (10 mg/kg ., 1x/day for 5 days), or treating female MaFIA mice 12 weeks partial meniscectomy (PMX) with AP20187 or vehicle control. We measured pain-related behaviors 1-3 days before and after depletion, and, 3-4 days after the last injection we examined joint histopathology and performed flow cytometry of the dorsal root ganglia (DRGs). In a separate cohort of male 8-week DMM mice or age-matched naïve vehicle controls, we conducted DRG bulk RNA-sequencing analyses after the 5-day vehicle or AP20187 treatment. Eight- and 16-weeks DMM in male mice, AP20187-induced macrophage depletion resulted in attenuated mechanical allodynia and knee hyperalgesia. Female mice showed alleviation of mechanical allodynia, knee hyperalgesia, and weight bearing deficits after macrophage depletion at 12 weeks PMX. Macrophage depletion did not affect the degree of cartilage degeneration, osteophyte width, or synovitis in either sex. Flow cytometry of the DRG revealed that macrophages and neutrophils were reduced after AP20187 treatment. In addition, in the DRG, only MHCII+ M1-like macrophages were significantly decreased, while CD163+MHCII- M2-like macrophages were not affected in both sexes. DRG bulk RNA-seq revealed that and were upregulated with DMM surgery compared to naïve mice, and downregulated in DMM after acute macrophage depletion. Acute systemic macrophage depletion reduced the levels of pro-inflammatory macrophages in the DRG and alleviated pain-related behaviors in established surgically induced OA in mice of both sexes, without affecting joint damage. Overall, these studies provide insight into immune cell regulation in the DRG during OA.

Objective: Osteoarthritis (OA) is a leading cause of chronic pain, yet OA pain management remains poor. Age is the strongest predictor of OA development, and mechanisms driving OA pain are unclear. While injury-induced OA models are useful, only a subset of OA is linked to traumatic injury. Here, we aimed to characterize age-associated joint damage, mechanical sensitization, and dorsal root ganglia (DRG) immune phenotypes in mice of both sexes. Methods: Male or female mice aged 6- or 20-months old were evaluated for histopathologic knee OA, pain-related behaviors, and L3-L5 dorsal root ganglia (DRG) immune characterization via flow cytometry. DRG gene expression in aged mice and humans was also examined. Results: Twenty-month old male mice had worse cartilage degeneration than 6-month old mice. Older female knees showed increased cartilage degeneration, but to a lesser degree than males. Older mice of both sexes had worse mechanical allodynia, knee hyperalgesia, and grip strength compared to younger mice. For both sexes, DRGs from older mice showed decreased CD45+ cells, and a significant increase in F4/80+ macrophages and CD11c+ dendritic cells. Older male DRGs showed increased expression of Ccl2 and Ccl5 and older female DRGs showed increased Cxcr4 and Ccl3 compared to 6-month DRGs, among other differentially expresssed genes. Human DRG analysis from six individuals >80 years old revealed elevated CCL2 in male DRGs compared to females, whereas CCL3 was higher in female DRGs. Conclusions: Here we show that aging in male and female mice is accompanied by mild knee OA, mechanical sensitization, and changes to immune cell populations in the DRG, suggesting novel avenues for development of analgesic therapies. Competing Interest Statement The following authors declare no conflicts of interest: TG, AMO, SI, MJW, JL, EBP, CRS, TMG and REM. AMM received consulting fees from Asahi Kasei Pharma Corporation, Eli Lilly, Pfizer, and Collegium Pharma.