Macrophage immunotherapy: overcoming impediments to realize promise.

As an essential component of immunity, macrophages have key roles in mammalian host defense, tissue homeostasis, and repair, as well as in disease pathogenesis and pathophysiology. A source of fascination and extensive research, in this Opinion we challenge the utility of the M1-M2 paradigm, and discuss the importance of accurate characterization of human macrophages. We comment on the application of single cell analytics to define macrophage subpopulations and how this could advance therapeutic options. We argue that human macrophage cell therapy can be used to alleviate many diseases, and offer a viewpoint on the knowledge gaps that must be filled to render such a therapeutic approach a reality and, ideally, a common future practice in precision medicine.

Vedolizumab: Potential Mechanisms of Action for Reducing Pathological Inflammation in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD), encompassing ulcerative colitis (UC), and Crohn's disease (CD), are a group of disorders characterized by chronic, relapsing, and remitting, or progressive inflammation along the gastrointestinal tract. IBD is accompanied by massive infiltration of circulating leukocytes into the intestinal mucosa. Leukocytes such as neutrophils, monocytes, and T-cells are recruited to the affected site, exacerbating inflammation and causing tissue damage. Current treatments used to block inflammation in IBD include aminosalicylates, corticosteroids, immunosuppressants, and biologics. The first successful biologic, which revolutionized IBD treatment, targeted the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Infliximab, adalimumab, and other anti-TNF antibodies neutralize TNFα, preventing interactions with its receptors and reducing the inflammatory response. However, up to 40% of people with IBD become unresponsive to anti-TNFα therapy. Thus, more recent biologics have been designed to block leukocyte trafficking to the inflamed intestine by targeting integrins and adhesins. For example, natalizumab targets the α4 chain of integrin heterodimers, α4ß1 and α4ß7, on leukocytes. However, binding of α4ß1 is associated with increased risk for developing progressive multifocal leukoencephalopathy, an often-fatal disease, and thus, it is not used to treat IBD. To target leukocyte infiltration without this life-threatening complication, vedolizumab was developed. Vedolizumab specifically targets the α4ß7 integrin and was approved to treat IBD based on the presumption that it would block T-cell recruitment to the intestine. Though vedolizumab is an effective treatment for IBD, some studies suggest that it may not block T-cell recruitment to the intestine and its mechanism(s) of action remain unclear. Vedolizumab may reduce inflammation by blocking recruitment of T-cells, or pro-inflammatory monocytes and dendritic cells to the intestine, and/or vedolizumab may lead to changes in the programming of innate and acquired immune cells dampening down inflammation.

COVID-19: Cytokine storm modulation/blockade with oral polyvalent immunoglobulins (PVIG, KMP01D): A potential and safe therapeutic agent (Primum nil nocere).

Although medication treatment in COVID-19 patients would have no direct effect on the spread of the disease, a shortening of the period of hospitalization by only a few days would release 25 - 30% of critical-care resources. However, there appears to be no well-established medication treatment available that can do this reliably at the present time. Anti-malarials currently being evaluated, i.e., chloroquine and hydroxychloroquine, are not yet established as effective medications, and antiviral agents, including remdesivir, are only weakly active. This position paper report is focused on the modulation of the cytokine storm since it appears to be a major cause of the multi-organ failure in COVID-19. Whereas corticosteroids are not recommended in patients not on mechanical ventilation, immunotherapy with convalescent plasma and intravenous immunoglobulin (IVIG) have been used with some success in COVID-19. There is emerging new evidence that polyvalent immunoglobulins (PVIG) from bovine colostrum given orally can also modulate the immune response. Research using lipopolysaccharide-stimulated peripheral blood mononuclear cells from colorectal cancer patients (a so called micro-cytokine storm) has shown that PVIG block the expression of pro-inflammatory cytokines and stimulate the expression of anti-inflammatory cytokines. We have been able to confirm these results in a similar model using mononuclear cells from healthy subjects and could demonstrate that the modulations produced by PVIG are quantitatively and qualitatively similar to those obtained using human immunoglobulin (IVIG). Both immunoglobulins reduce the lipopolysaccharide-induced increase in inflammatory cytokines, interleukin (IL-) 12/23p40 (-90%), IL-6 (-75%) and TNF-α (-60%) and increased the levels of the anti-inflammatory cytokine, IL-10 (+75%). Evidence is presented that PVIG can produce anti-inflammatory effects similar to these after oral application in patients. Its use is contraindicated in patients with lactose intolerance but is otherwise safe and free of complications in clinical studies including the treatment of infants with gastrointestinal disorders. Conclusion: PVIG appears to be a potential and safe anti-inflammatory agent and can be recommended as a candidate medication for studies in COVID-19 patients.

Gut microbes in pediatric ALL survivorship.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and has a 5 year survival rate of greater than 90%. Despite this extraordinary success, survivors face lifelong chronic health problems including a predisposition to obesity, metabolic syndrome, and resulting complications like cardiovascular disease. In this issue, Thomas et al. (Yang laboratory) investigated the gut microbiome in pediatric ALL survivors and healthy sibling controls. They identified key changes in operational taxonomic units (OTUs), which have been linked previously to obesity and metabolic syndrome. This study suggests that dysbiosis, which can predispose to life-long secondary complications of ALL, begins in childhood immediately after treatment and opens an ample window for interventions aimed at reducing obesity and metabolic syndrome in ALL survivors.

FcRn is a CD32a coreceptor that determines susceptibility to IgG immune complex-driven autoimmunity.

IgG immune complexes (ICs) promote autoimmunity through binding fragment crystallizable (Fc) γ-receptors (FcγRs). Of these, the highly prevalent FcγRIIa (CD32a) histidine (H)-131 variant (CD32aH) is strongly linked to human autoimmune diseases through unclear mechanisms. We show that, relative to the CD32a arginine (R)-131 (CD32aR) variant, CD32aH more avidly bound human (h) IgG1 IC and formed a ternary complex with the neonatal Fc receptor (FcRn) under acidic conditions. In primary human and mouse cells, both CD32a variants required FcRn to induce innate and adaptive immune responses to hIgG1 ICs, which were augmented in the setting of CD32aH. Conversely, FcRn induced responses to IgG IC independently of classical FcγR, but optimal responses required FcRn and FcγR. Finally, FcRn blockade decreased inflammation in a rheumatoid arthritis model without reducing circulating autoantibody levels, providing support for FcRn's direct role in IgG IC-associated inflammation. Thus, CD32a and FcRn coregulate IgG IC-mediated immunity in a manner favoring the CD32aH variant, providing a novel mechanism for its disease association.

How do immune and mesenchymal cells influence the intestinal epithelial cell compartment in inflammatory bowel disease? Let's crosstalk about it!

Intestinal epithelial cells provide a front line of defense by establishing a barrier against food Ags, pathogens, and commensal microorganisms. This defense includes the establishment of a tolerogenic environment in the gastrointestinal (GI) tract. The intestinal epithelium replenishes itself by cell turnover every 4-5 days, and this process is facilitated by various pathways of communication between the intestinal epithelial cells (IECs), the underlying stromal cell network, and professional immune cells, which together help establish a proper intestinal stem cell (ISC) niche in the crypt. However, during a state of inflammation, such as in inflammatory bowel diseases (IBD), these communication pathways can be altered, and this can lead to the development of inflammatory IECs within the crypt that further drive inflammation. Here, we review the current literature looking at crosstalk between immune cells, stromal cells, and IECs: how does the immune system potentially alter the ISC niche, and how do IECs influence intestinal immunity? We discuss the latest research using single cell RNA sequencing and intestinal organoid cultures to help answer these questions. A better understanding of this complex crosstalk can help lead to a better understanding of intestinal biology in general, and more efficient therapeutic approaches to treat IBD.

Enteroids Derived From Inflammatory Bowel Disease Patients Display Dysregulated Endoplasmic Reticulum Stress Pathways, Leading to Differential Inflammatory Responses and Dendritic Cell Maturation.

BACKGROUND AND AIMS: Endoplasmic reticulum [ER] stress in intestinal epithelial cells [IECs] contributes to the pathogenesis of inflammatory bowel disease [IBD]. We hypothesized that ER stress changes innate signalling in human IECs, augmenting toll-like receptor [TLR] responses and inducing pro-inflammatory changes in underlying dendritic cells [DCs]. METHODS: Caco-2 cells and primary human colon-derived enteroid monolayers were exposed to ATP [control stressor] or thapsigargin [Tg] [ER stress inducer], and were stimulated with the TLR5 agonist flagellin. Cytokine release was measured by an enzyme immunoassay. ER stress markers CHOP, GRP78 and XBP1s/u were measured via quantitative PCR and Western blot. Monocyte-derived DCs [moDCs] were cultured with the IEC supernatants and their activation state was measured. Responses from enteroids derived from IBD patients and healthy control participants were compared. RESULTS: ER stress enhanced flagellin-induced IL-8 release from Caco-2 cells and enteroids. Moreover, conditioned media activated DCs to become pro-inflammatory, with increased expression of CD80, CD86, MHCII, IL-6, IL-15 and IL-12p70 and decreased expression of CD103 and IL-10. Flagellin-induced IL-8 production correlated with DC activation, suggesting a common stress pathway. Moreover, there were distinct differences in cytokine expression and basal ER stress between IBD and healthy subject-derived enteroid monolayers, suggesting a dysregulated ER stress pathway in IBD-derived enteroids. CONCLUSIONS: Cellular stress enhances TLR5 responses in IECs, leading to increased DC activation, indicating a previously unknown mechanistic link between epithelial ER stress and immune activation in IBD. Furthermore, dysregulated ER stress may be propagated from the intestinal epithelial stem cell niche in IBD patients.

Malt1 deficient mice develop osteoporosis independent of osteoclast-intrinsic effects of Malt1 deficiency.

This study tested the hypothesis that mucosa associated lymphoid tissue 1 (Malt1) deficiency causes osteoporosis in mice by increasing osteoclastogenesis and osteoclast activity. A patient with combined immunodeficiency (CID) caused by MALT1 deficiency had low bone mineral density resulting in multiple low impact fractures that was corrected by hematopoietic stem cell transplant (HSCT). We have reported that Malt1 deficient Mϕs, another myeloid cell type, are hyper-responsive to inflammatory stimuli. Our objectives were to determine whether Malt1 deficient mice develop an osteoporosis-like phenotype and whether it was caused by Malt1 deficiency in osteoclasts. We found that Malt1 deficient mice had low bone volume by 12 weeks of age, which was primarily associated with reduced trabecular bone. Malt1 protein is expressed and active in osteoclasts and is induced by receptor activator of NF-κB ligand (RANKL) in preosteoclasts. Malt1 deficiency did not impact osteoclast differentiation or activity in vitro. However, Malt1 deficient (Malt1-/- ) mice had more osteoclasts in vivo and had lower levels of serum osteoprotegerin (OPG), an endogenous inhibitor of osteoclastogenesis. Inhibition of Malt1 activity in Mϕs induced MCSF production, required for osteoclastogenesis, and decreased OPG production in response to inflammatory stimuli. In vitro, MCSF increased and OPG inhibited osteoclastogenesis, but effects were not enhanced in Malt1 deficient osteoclasts. These data support the hypothesis that Malt1 deficient mice develop an osteoporotic phenotype with increased osteoclastogenesis in vivo, but suggest that this is caused by inflammation rather than an effect of Malt1 deficiency in osteoclasts.

Phosphatidylinositol 3-kinase p110δ drives intestinal fibrosis in SHIP deficiency.

Crohn's disease is an immune-mediated disease characterized by inflammation along the gastrointestinal tract. Fibrosis requiring surgery occurs in one-third of people with Crohn's disease but there are no treatments for intestinal fibrosis. Mice deficient in the SH2 domain-containing inositolpolyphosphate 5'-phosphatase (SHIP), a negative regulator of phosphatidylinositol 3-kinase (PI3K) develop spontaneous Crohn's disease-like intestinal inflammation and arginase I (argI)-dependent fibrosis. ArgI is up-regulated in SHIP deficiency by PI3Kp110δ activity. Thus, we hypothesized that SHIP-deficient mice develop fibrosis due to increased PI3Kp110δ activity. In SHIP-deficient mice, genetic ablation or pharmacological inhibition of PI3Kp110δ activity reduced intestinal fibrosis, including muscle thickening, accumulation of vimentin+ mesenchymal cells, and collagen deposition. PI3Kp110δ deficiency or inhibition also reduced ileal inflammation in SHIP-deficient mice suggesting that PI3Kp110δ may contribute to inflammation. Targeting PI3Kp110δ activity may be an effective strategy to reduce intestinal fibrosis, and may be particularly effective in the subset of people with Crohn's disease, who have low SHIP activity.

Integrins and ERp57 Coordinate to Regulate Cell Surface Calreticulin in Immunogenic Cell Death.

Therapy-induced presentation of cell surface calreticulin (CRT) is a pro-phagocytic immunogen beneficial for invoking anti-tumor immunity. Here, we characterized the roles of ERp57 and α-integrins as CRT-interacting proteins that coordinately regulate CRT translocation from the ER to the surface during immunogenic cell death. Using T-lymphoblasts as a genetic cell model, we found that drug-induced surface CRT is dependent on ERp57, while drug-induced surface ERp57 is independent of CRT. Differential subcellular immunostaining assays revealed that ERp57-/- cells have minimal cytosolic CRT, indicating that ERp57 is indispensable for extra-ER accumulation of CRT. Stimulation of integrin activity, with either cell adhesion or molecular agonists, resulted in decreased drug-induced surface CRT and ERp57 levels. Similarly, surface CRT and ERp57 was reduced in cells expressing GFFKR, a conserved α-integrin cytosolic motif that binds CRT. Drug-induced surface ERp57 levels were consistently higher in CRT-/- cells, suggesting integrin inhibition of surface ERp57 is an indirect consequence of α-integrin binding to CRT within the CRT-ERp57 complex. Furthermore, ß1-/- cells with reduced expression of multiple α-integrins, exhibit enhanced levels of drug-induced surface CRT and ERp57. Our findings highlight the coordinate involvement of plasma membrane integrins as inhibitors, and ERp57 originating from the ER as promoters, of CRT translocation from the ER to the cell surface.

Intravenous immunoglobulin (IVIg) or IVIg-treated macrophages reduce DSS-induced colitis by inducing macrophage IL-10 production.

Intravenous immunoglobulin (IVIg) is used to treat immune-mediated diseases but its mechanism of action is poorly understood. We have reported that co-treatment with IVIg and lipopolysaccharide activates macrophages to produce large amounts of anti-inflammatory IL-10 in vitro. Thus, we asked whether IVIg-treated macrophages or IVIg could reduce intestinal inflammation in mice during dextran sulfate sodium (DSS)-induced colitis by inducing macrophage IL-10 production in vivo. Adoptive transfer of IVIg-treated macrophages reduces intestinal inflammation in mice and collagen accumulation post-DSS. IVIg treatment also reduces DSS-induced intestinal inflammation and its activity is dependent on the Fc portion of the antibody. Ex vivo, IVIg induces IL-10 production and reduces IL-12/23p40 and IL-1ß production in colon explant cultures. Co-staining tissues for mRNA, we demonstrate that macrophages are the source of IL-10 in IVIg-treated mice; and using IL-10-GFP reporter mice, we demonstrate that IVIg induces IL-10 production by intestinal macrophages. Finally, IVIg-mediated protection is lost in mice deficient in macrophage IL-10 production (LysMcre+/- IL-10fl/fl mice). Together, our data demonstrate a novel, in vivo mechanism of action for IVIg. IVIg-treated macrophages or IVIg could be used to treat people with intestinal inflammation and may be particularly useful for people with inflammatory bowel disease, who are refractory to therapy.

Depletion and Reconstitution of Macrophages in Mice.

Macrophages are innate immune cells, which have important roles in the inflammatory response to infections or tissue injury, and have an equally important role in the resolution of inflammation. Macrophages play a key part in directing the innate immune response and subsequent adaptive immune response. They can acquire a variety of distinct but also overlapping activation states, depending on the local microenvironment, in order to perform these functions. Stimuli, such as IFNγ and LPS, can promote an inflammatory activation state, which is associated with the production of reactive oxygen species, and pro-inflammatory cytokines and chemokines. Immune complexes and LPS can promote an anti-inflammatory activation state to prevent damage to the host, which is associated with the production of high levels of the anti-inflammatory cytokine IL-10 and low levels of pro-inflammatory cytokines. Wound-healing macrophages can be activated by IL-4 or IL-13 and have roles in tissue remodeling and the resolution of inflammation. Macrophages are present in nearly every tissue of the body and are important for maintaining homeostasis, but their dysfunction can also lead to diseases, such as inflammatory bowel disease. To study the role macrophages play in a complex in vivo environment, depletion and reconstitution experiments can be utilized. Clodronate liposomes are an effective and versatile way to deplete macrophages in vivo; they can allow selective depletion from tissues of interest and can be used on transgenic mice. However, clodronate liposomes deplete all types of macrophages as well as dendritic cells, so other strategies are required in parallel to determine whether macrophages or macrophages of a particular activation state are required. Reconstitution of macrophages by adoptive transfer can be performed, with or without prior depletion, to further suggest that the observed effect is macrophage dependent. Macrophages activated ex vivo or macrophages from transgenic mice can be adoptively transferred during disease models to determine whether a specific protein or activation state affects disease outcome. Macrophage contribution to health and disease can be effectively studied using depletion with clodronate liposomes and by macrophage reconstitution, as demonstrated in this chapter.

IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant.

Intravenous Immunoglobulin (IVIg) is used to treat autoimmune or inflammatory diseases, but its mechanism of action is not completely understood. We asked whether IVIg can induce interleukin-10 (IL-10) and reduce pro-inflammatory cytokine production in human monocytes, and whether this response is reduced in monocytes from people with an Fcγ receptor IIA (FcγRIIA) gene variant, which is associated with increased risk of inflammatory diseases and poor response to antibody-based biological therapy. IVIg increased IL-10 production and reduced pro-inflammatory cytokine production in response to bacterial lipopolysaccharide (LPS), which required FcγRI and FcγRIIB and activation of MAPKs, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38. IL-10 production was lower and pro-inflammatory cytokine production was higher in monocytes from people with the FcγRIIA risk variant and the risk variant prevented IL-10 production in response to (IVIg+LPS). Finally, we show that IVIg did not induce MAPK activation in monocytes from people with the risk variant. Our results demonstrate that IVIg can skew human monocytes to an anti-inflammatory, IL-10-producing activation state, which is compromised in monocytes from people with the FcγRIIA risk variant. This research has profound implications for the use of IVIg because 25% of the population is homozygous for the FcγRIIA risk variant and its efficacy may be reduced in those individuals. In addition, this research may be useful to develop new therapeutic strategies to replace IVIg by cross-linking FcγRIs and FcγRIIBs to promote anti-inflammatory macrophage activation, independent of the FcγRIIA genotype.

Malt1 blocks IL-1ß production by macrophages in vitro and limits dextran sodium sulfate-induced intestinal inflammation in vivo.

This study tested the hypothesis that Malt1 deficiency in macrophages contributes to dextran sodium sulfate (DSS)-induced intestinal inflammation in Malt1-deficient mice. In people, combined immunodeficiency caused by a homozygous mutation in the MALT1 gene is associated with increased susceptibility to bacterial infections and chronic inflammation, including severe inflammation along the gastrointestinal tract. The consequences of Malt1 deficiency have largely been attributed to its role in lymphocytes, but Malt1 is also expressed in macrophages, where it is activated downstream of TLR4 and dectin-1. The effect of Malt1 deficiency in murine macrophages and its contribution to DSS-induced colitis have not been investigated. Our objectives were to compare the susceptibility of Malt1+/+ and Malt1-/- mice to DSS-induced colitis, to determine the contribution of macrophages to DSS-induced colitis in Malt1-/- mice, and to assess the effect of innate immune stimuli on Malt1-/- macrophage inflammatory responses. We found that Malt1 deficiency exacerbates DSS-induced colitis in mice, accompanied by higher levels of IL-1ß, and that macrophages and IL-1 signaling contribute to pathology in Malt1-/- mice. Malt1-/- macrophages produce more IL-1ß in response to either TLR4 or dectin-1 ligation, whereas inhibition of Malt1 proteolytic (paracaspase) activity blocked IL-1ß production. TLR4 or dectin-1 stimulation induced Malt1 protein levels but decreased its paracaspase activity. Taken together, these data support the hypothesis that Malt1-/- macrophages contribute to increased susceptibility of Malt1-/- mice to DSS-induced colitis, which is dependent on IL-1 signaling. Increased IL-1ß production by MALT1-deficient macrophages may also contribute to chronic inflammation in people deficient in MALT1.

CD47-ligation induced cell death in T-acute lymphoblastic leukemia.

CD47 is a cell-surface marker well recognized for its anti-phagocytic functions. As such, an emerging avenue for targeted cancer therapies involves neutralizing the anti-phagocytic function using monoclonal antibodies (mAbs) to enhance tumour cell immunogenicity. A lesser known consequence of CD47 receptor ligation is the direct induction of tumour cell death. While several mAbs and their derivatives with this property have been studied, the best characterized is the commercially available mAb B6H12, which requires immobilization for induction of cell death. Here, we describe a commercially available mAb, CC2C6, which induces T-cell acute lymphoblastic leukemia (ALL) cell death in soluble form. Soluble CC2C6 induces CD47-dependent cell death in a manner consistent with immobilized B6H12, which is characterized by mitochondrial deficiencies but is independent of caspase activation. Titration studies indicated that CC2C6 shares a common CD47-epitope with B6H12. Importantly, CC2C6 retains the anti-phagocytic neutralizing function, thus possessing dual anti-tumour properties. Although CD47-ligation induced cell death occurs in a caspase-independent manner, CC2C6 was found to stimulate increases in Mcl-1 and NOXA levels, two Bcl-2 family proteins that govern the intrinsic apoptosis pathway. Further analysis revealed that the ratio of Mcl-1:NOXA were minimally altered for cells treated with CC2C6, in comparison to cells treated with agents that induced caspase-dependent apoptosis which alter this ratio in favour of NOXA. Finally, we found that CC2C6 can synergize with low dose chemotherapeutic agents that induce classical apoptosis, giving rise to the possibility of an effective combination treatment with reduced long-term sequelae associated with high-dose chemotherapies in childhood ALL.

Inflammatory bowel disease and immunonutrition: novel therapeutic approaches through modulation of diet and the gut microbiome.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, thought to at least in part reflect an aberrant immune response to gut bacteria. IBD is increasing in incidence, particularly in populations that have recently immigrated to western countries. This suggests that environmental factors are involved in its pathogenesis. We hypothesize that the increase in IBD rates might reflect the consumption of an unhealthy Western diet, containing excess calories and lacking in key nutritional factors, such as fibre and vitamin D. Several recent studies have determined that dietary factors can dramatically influence the activation of immune cells and the mediators they release through a process called immunonutrition. Moreover, dietary changes can profoundly affect the balance of beneficial versus pathogenic bacteria in the gut. This microbial imbalance can alter levels of microbiota-derived metabolites that in turn can influence innate and adaptive intestinal immune responses. If the diet-gut microbiome disease axis does indeed underpin much of the 'western' influence on the onset and progression of IBD, then tremendous opportunity exists for therapeutic changes in lifestyle, to modulate the gut microbiome and to correct immune imbalances in individuals with IBD. This review highlights four such therapeutic strategies - probiotics, prebiotics, vitamin D and caloric restriction - that have the potential to improve and add to current IBD treatment regimens.

SHIP negatively regulates type II immune responses in mast cells and macrophages.

SHIP is a hematopoietic-specific lipid phosphatase that dephosphorylates PI3K-generated PI(3,4,5)-trisphosphate. SHIP removes this second messenger from the cell membrane blunting PI3K activity in immune cells. Thus, SHIP negatively regulates mast cell activation downstream of multiple receptors. SHIP has been referred to as the "gatekeeper" of mast cell degranulation as loss of SHIP dramatically increases degranulation or permits degranulation in response to normally inert stimuli. SHIP also negatively regulates Mϕ activation, including both pro-inflammatory cytokine production downstream of pattern recognition receptors, and alternative Mϕ activation by the type II cytokines, IL-4, and IL-13. In the SHIP-deficient (SHIP-/- ) mouse, increased mast cell and Mϕ activation leads to spontaneous inflammatory pathology at mucosal sites, which is characterized by high levels of type II inflammatory cytokines. SHIP-/- mast cells and Mϕs have both been implicated in driving inflammation in the SHIP-/- mouse lung. SHIP-/- Mϕs drive Crohn's disease-like intestinal inflammation and fibrosis, which is dependent on heightened responses to innate immune stimuli generating IL-1, and IL-4 inducing abundant arginase I. Both lung and gut pathology translate to human disease as low SHIP levels and activity have been associated with allergy and with Crohn's disease in people. In this review, we summarize seminal literature and recent advances that provide insight into SHIP's role in mast cells and Mϕs, the contribution of these cell types to pathology in the SHIP-/- mouse, and describe how these findings translate to human disease and potential therapies.

IFN-γ directly inhibits murine B-cell precursor leukemia-initiating cell proliferation early in life.

The early-life immune environment has been implicated as a modulator of acute lymphoblastic leukemia (ALL) development in children, with infection being associated with significant changes in ALL risk. Furthermore, polymorphisms in several cytokine genes, including IL-10 and IFN-γ, are associated with leukemia development. However, the mechanisms and timing of these influences remain unknown. Here, we use the Eµ-ret transgenic mouse model of B-cell precursor ALL to assess the influence of IFN-γ on the early-life burden of leukemia-initiating cells. The absence of IFN-γ activity resulted in greater numbers of leukemia-initiating cells early in life and was associated with accelerated leukemia onset. The leukemia-initiating cells from IFN-γ-knockout mice had reduced suppressor of cytokine signaling (SOCS-1) expression, were significantly more sensitive to IFN-γ, and exhibited more rapid expansion in vivo than their wild-type counterparts. However, sensitivity to this inhibitory pathway was lost in fully transformed IFN-γ-knockout leukemia cells. These results demonstrate that the influence of IFN-γ on ALL progression may not be mediated by selection of nascent transformed cells but rather through a general SOCS-mediated reduction in B-cell precursor proliferation. Thus, while cytokine levels may influence leukemia at multiple points during disease progression, our study indicates a significant early influence of basal, infection-independent cytokine production on leukemogenesis.

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNFα) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Endosomal pH modulation by peptide-gold nanoparticle hybrids enables potent anti-inflammatory activity in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays a central role in the pathophysiology of many acute and chronic human inflammatory diseases, and pharmacological regulation of TLR responses is anticipated to be beneficial in many inflammatory conditions. Currently there are no specific TLR inhibitors in clinical use. To overcome this challenge, we have developed a nano-based TLR inhibitor (peptide-gold nanoparticle hybrids) that inhibits a broad spectrum of TLR responses. Through mechanistic studies, we established that specific peptide decorated-gold nanoparticles that display high cellular uptake in phagocytic immune cells modulate endosomal pH, leading to significant attenuation of signaling through multiple TLRs. Using a global transcriptomic approach, we defined the broad anti-inflammatory activity of the nanoparticle in human peripheral blood mononuclear cells. In vivo studies confirmed the beneficial immunomodulatory activity since treatment with the nanoparticle significantly reduced weight loss, improved the disease activity index, and ameliorated colonic inflammation in a murine model of intestinal inflammation. This work enhances our fundamental understanding of the role of peptide coatings on the nanoparticle surface in regulating innate immune signaling, and identifies specific peptide decorated nanoparticles that may represent a novel class of anti-inflammatory therapeutics for human inflammatory diseases.