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.

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.

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.

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.

Activity of SHIP, Which Prevents Expression of Interleukin 1ß, Is Reduced in Patients With Crohn's Disease.

BACKGROUND & AIMS: Crohn's disease (CD) is associated with a dysregulated immune response to commensal micro-organisms in the intestine. Mice deficient in inositol polyphosphate 5'-phosphatase D (INPP5D, also known as SHIP) develop intestinal inflammation resembling that of patients with CD. SHIP is a negative regulator of PI3Kp110α activity. We investigated mechanisms of intestinal inflammation in Inpp5d(-/-) mice (SHIP-null mice), and SHIP levels and activity in intestinal tissues of subjects with CD. METHODS: We collected intestines from SHIP-null mice, as well as Inpp5d(+/+) mice (controls), and measured levels of cytokines of the interleukin 1 (IL1) family (IL1α, IL1ß, IL1ra, and IL6) by enzyme-linked immunosorbent assay. Macrophages were isolated from lamina propria cells of mice, IL1ß production was measured, and mechanisms of increased IL1ß production were investigated. Macrophages were incubated with pan-phosphatidylinositol 3-kinase inhibitors or PI3Kp110α-specific inhibitors. Some mice were given an antagonist of the IL1 receptor; macrophages were depleted from ilea of mice using clodronate-containing liposomes. We obtained ileal biopsies from sites of inflammation and peripheral blood mononuclear cells (PBMCs) from treatment-naïve subjects with CD or without CD (controls), and measured SHIP levels and activity. PBMCs were incubated with lipopolysaccharide and adenosine triphosphate, and levels of IL1ß production were measured. RESULTS: Inflamed intestinal tissues and intestinal macrophages from SHIP-null mice produced higher levels of IL1B and IL18 than intestinal tissues from control mice. We found PI3Kp110α to be required for macrophage transcription of Il1b. Macrophage depletion or injection of an IL1 receptor antagonist reduced ileal inflammation in SHIP-null mice. Inflamed ileal tissues and PBMCs from patients with CD had lower levels of SHIP protein than controls (P < .0001 and P < .0002, respectively). There was an inverse correlation between levels of SHIP activity in PBMCs and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate (R(2) = .88). CONCLUSIONS: Macrophages from SHIP-deficient mice have increased PI3Kp110α-mediated transcription of Il1b, which contributes to spontaneous ileal inflammation. SHIP levels and activity are lower in intestinal tissues and peripheral blood samples from patients with CD than controls. There is an inverse correlation between SHIP activity and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate in PBMCs. Strategies to reduce IL1B might be developed to treat patients with CD found to have low SHIP activity.

Intravenous immunoglobulin skews macrophages to an anti-inflammatory, IL-10-producing activation state.

Intravenous Ig is used to treat autoimmune or autoinflammatory disorders, but the mechanism by which it exerts its immunosuppressive activity is not understood completely. To examine the impact of intravenous Ig on macrophages, we compared cytokine production by LPS-activated macrophages in the presence and absence of intravenous Ig. Intravenous Ig treatment induced robust production of IL-10 in response to LPS, relative to LPS stimulation alone, and reduced production of proinflammatory cytokines. This anti-inflammatory, intravenous Ig-induced activation was sustained for 24 h but could only be induced if intravenous Ig were provided within 1 h of LPS stimulation. Intravenous Ig activation led to enhanced and prolonged activation of MAPKs, Erk1/2, p38, and Erk5, and inhibition of each reduced intravenous Ig-induced IL-10 production and suppression of IL-12/23p40. IL-10 production occurred rapidly in response to intravenous Ig + LPS and was sufficient to reduce proinflammatory IL-12/23p40 production in response to LPS. IL-10 induction and reduced IL-12/23p40 production were transcriptionally regulated. IL-10 played a direct role in reducing proinflammatory cytokine production by macrophages treated with intravenous Ig + LPS, as macrophages from mice deficient in the IL-10R ß chain or in IL-10 were compromised in their ability to reduce proinflammatory cytokine production. Finally, intraperitoneal injection of intravenous Ig or intravenous Ig + LPS into mice activated macrophages to produce high levels of IL-10 during subsequent or concurrent LPS challenge, respectively. These findings identify IL-10 as a key anti-inflammatory mediator produced by intravenous Ig-treated macrophages and provide insight into a novel mechanism by which intravenous Ig may dampen down inflammatory responses in patients with autoimmune or autoinflammatory diseases.

Arginase activity in alternatively activated macrophages protects PI3Kp110δ deficient mice from dextran sodium sulfate induced intestinal inflammation.

Alternatively activated or M2 macrophages have been reported to protect mice from intestinal inflammation, but the mechanism of protection has not been elucidated. In this study, we demonstrate that mice deficient in the p110δ catalytic subunit activity of class I phosphatidylinositol 3-kinase (PI3Kp110δ) have increased clinical disease activity and histological damage during dextran sodium sulfate (DSS) induced colitis. Increased disease severity in PI3Kp110δ-deficient mice is dependent on professional phagocytes and correlates with reduced numbers of arginase I+ M2 macrophages in the colon and increased production of inflammatory nitric oxide. We further demonstrate that PI3Kp110δ-deficient macrophages are defective in their ability to induce arginase I when skewed to an M2 phenotype with IL-4. Importantly, adoptive transfer of IL-4-treated macrophages derived from WT mice, but not those from PI3Kp110δ-deficient mice, protects mice during DSS-induced colitis. Moreover, M2 macrophages mediated protection is lost when mice are cotreated with inhibitors that block arginase activity or during adoptive transfer of arginase I deficient M2 macrophages. Taken together, our data demonstrate that arginase I activity is required for M2 macrophages mediated protection during DSS-induced colitis in PI3Kp110δ-deficient mice.