Single particle tracking (SPT) is often the rate-limiting step in live cell imaging studies of sub-cellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout the movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long and short-lived endocytic structures, and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.

Toll-like receptors (TLRs) recognize microbial components and trigger the inflammatory and immune responses against pathogens. IkappaBzeta (also known as MAIL and INAP) is an ankyrin-repeat-containing nuclear protein that is highly homologous to the IkappaB family member Bcl-3 (refs 1-6). Transcription of IkappaBzeta is rapidly induced by stimulation with TLR ligands and interleukin-1 (IL-1). Here we show that IkappaBzeta is indispensable for the expression of a subset of genes activated in TLR/IL-1R signalling pathways. IkappaBzeta-deficient cells show severe impairment of IL-6 production in response to a variety of TLR ligands as well as IL-1, but not in response to tumour-necrosis factor-alpha. Endogenous IkappaBzeta specifically associates with the p50 subunit of NF-kappaB, and is recruited to the NF-kappaB binding site of the IL-6 promoter on stimulation. Moreover, NF-kappaB1/p50-deficient mice show responses to TLR/IL-1R ligands similar to those of IkappaBzeta-deficient mice. Endotoxin-induced expression of other genes such as Il12b and Csf2 is also abrogated in IkappaBzeta-deficient macrophages. Given that the lipopolysaccharide-induced transcription of IkappaBzeta occurs earlier than transcription of these genes, some TLR/IL-1R-mediated responses may be regulated in a gene expression process of at least two steps that requires inducible IkappaBzeta.

Salmonella sneaks past security C ertain gut cells can leave resident bacteria alone but respond selectively to invaders. Satoshi Uematsu, Shizuo Akira, and colleagues (Osaka University, Japan) suggest that gut cells achieve this differentiation by using a special, pathogen-specifi c receptor called the Toll-like receptor 5 (TLR5). But the pathogenic Salmonella typhimurium turns the situation around: events triggered by the special receptor help the bug to invade its host. TLRs, which are expressed on professional antigen-presenting cells, recognize common pathogen-associated molecules and trigger innate immunity. TLR5 on dendritic cells recognizes bacterial fl agellin protein in vitro, but its function in vivo was previously unknown. Akira's team found that TLR5 mRNA was highly expressed in the mouse intestine particularly in a specifi c subpopulation of antigen-presenting lamina propria cells (CD11c + LPCs). In these cells, TLR5 was necessary for bacterial fl agellin to induce infl ammatory cytokines, yet when the team infected TLR5 −/− mice with Salmonella, a fl agellated bacterium, these mice were unexpectedly resistant to the bug. It was not, however, invasion of the CD11c + LPCs that showed a difference. In the gut, Salmonella invaded the CD11c + LPCs in both TLR5 +/+ and TLR5 −/− mice. However, in the TLR5 −/− mice, fewer bacteria-laden CD11c + LPCs moved from the intestinal tract to the mesenteric lymph nodes, probably because the LPCs failed to be activated by the bacteria. These mice had more resistance to systemic infection-fewer bacteria reached their livers and spleens-but it is not yet clear whether a similar tactic of TLR5 blocking would work in humans.

Summary The mechanisms that govern receptor coalescence into functional clusters – often a critical step in their stimulation by ligand – are poorly understood. We used single-molecule tracking to investigate the dynamics of CD36, a clustering-responsive receptor that mediates oxidized LDL uptake by macrophages. We found that CD36 motion in the membrane was spatially structured by the cortical cytoskeleton. A subpopulation of receptors diffused within linear confinement regions that simultaneously facilitated freedom of movement along one axis while increasing the effective receptor density. Co-confinement within troughs enhanced the probability of collisions between unligated receptors and promoted their clustering. Cytoskeleton perturbations that inhibited diffusion in linear confinement regions reduced receptor clustering in the absence of ligand, and, following ligand addition, suppressed CD36-mediated signaling and internalization. These observations demonstrate a role for the cytoskeleton in controlling signal transduction by structuring receptor diffusion within membrane regions that increase their collision frequency.

Interleukin-10 (IL-10) plays an important role in prevention of chronic inflammation in vivo. However, the molecular mechanism by which IL-10 exerts its antiinflammatory response is poorly understood. Here, we performed a microarray analysis and identified Bcl-3 as an IL-10-inducible gene in macrophages. Lentiviral vector-mediated expression of Bcl-3 inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor ␣ (TNF-␣), but not IL-6, in macrophages. In Bcl-3-transduced and IL-10-pretreated macrophages, LPS-induced nuclear translocation of nuclear factor B (NF-B) p65 was not impaired. However, DNA binding by NF-B p50/p65 was profoundly inhibited. Nuclear localization of Bcl-3 was associated with inhibition of LPS-induced TNF-␣ production. Overexpression of Bcl-3 suppressed activation of the TNF-␣ promoter, but not the IL-6 promoter. Bcl-3 interacted with NF-B p50 and was recruited to the TNF-␣ promoter, but not the IL-6 promoter, indicating that Bcl-3 facilitates p50-mediated inhibition of TNF-␣ expression. Furthermore, Bcl-3-deficient macrophages showed defective IL-10-mediated suppression of LPS induction of TNF-␣, but not IL-6. These findings suggest that IL-10-induced Bcl-3 is required for suppression of TNF-␣ production in macrophages. IntroductionInterleukin-10 (IL-10) is produced by activated macrophages and T cells, and plays an important role in anti-inflammatory responses. 1 Indeed, mice lacking IL-10 or the IL-10 receptor developed chronic enterocolitis and showed enhanced inflammatory responses. [2][3][4] Furthermore, transfer of the IL-10 gene reduced the incidence of colitis in mice, and treatment with IL-10 is now anticipated to be used clinically for patients with inflammatory bowel diseases. 5,6 The anti-inflammatory activity of IL-10 is mainly elicited by inhibition of the activity of macrophages/monocytes. IL-10 inhibits lipopolysaccharide (LPS)-induced production of inflammatory cytokines, including tumor necrosis factor ␣ (TNF-␣), IL-6, and IL-12, by macrophages. Many attempts have been made to elucidate the mechanism by which IL-10 inhibits cytokine production by macrophages, and several models have been proposed. These include inhibition of nuclear factor B (NF-B) and mitogen-activated protein (MAP) kinase activity, and reduction of transcription and stability of mRNA for cytokine genes. [7][8][9][10] However, the precise mechanism by which IL-10 suppresses macrophage activity remains unclear.The IL-10 receptor consists of the ligand binding subunit, IL-10R1, and an accessory subunit, IL-10R2. Binding of IL-10 to the IL-10 receptor induces activation of the Janus kinase (JAK) family of tyrosine kinases, Jak1 and Tyk2, which are constitutively associated with IL-10R1 and IL-10R2, respectively. 1 Jak1 and Tyk2 activation is followed by phosphorylation of the latent transcription factor signal transducer and activator of transcription 3 (Stat3). Studies with gene-targeted mice have revealed that Jak1 and Stat3 play a pivotal role in the IL-10 signaling pathway. Macrophages ...

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMφ). CLPMφ of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMφ gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMφ. These genes included nuclear IκB proteins such as Bcl-3 and IκBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-α production, we analyzed the role of IκBNS in macrophages. Lentiviral introduction of IκBNS resulted in impaired LPS-induced IL-6 production, but not TNF-α production in the murine macrophage cell line RAW264.7. IκBNS expression led to constitutive and intense DNA binding of NF-κB p50/p50 homodimers. IκBNS was recruited to the IL-6 promoter, but not to the TNF-α promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IκBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-α. Thus, IκBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IκB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.

Toll-like receptor (TLR)-mediated immune responses are downregulated by several mechanisms that affect signaling pathways. However, it remains elusive how TLR-mediated gene expression is differentially modulated. Here, we show that IkappaBNS, a TLR-inducible nuclear IkappaB protein, negatively regulates induction of a subset of TLR-dependent genes through inhibition of NF-kappaB activity. IkappaBNS-deficient macrophages and dendritic cells show increased TLR-mediated expression of genes such as IL-6 and IL-12p40, which are induced late after TLR stimulation. In contrast, IkappaBNS-deficient cells showed normal induction of genes that are induced early or induced via IRF-3 activation. LPS stimulation of IkappaBNS-deficient macrophages prolonged NF-kappaB activity at the specific promoters, indicating that IkappaBNS mediates termination of NF-kappaB activity at selective gene promoters. Moreover, IkappaBNS-deficient mice are highly susceptible to LPS-induced endotoxin shock and intestinal inflammation. Thus, IkappaBNS regulates inflammatory responses by inhibiting the induction of a subset of TLR-dependent genes through modulation of NF-kappaB activity.

Mycobacterium tuberculosis invades alveolar epithelial cells as well as macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we report that lipocalin 2 (Lcn2)-dependent inhibition of mycobacterial growth within epithelial cells is required for anti-mycobacterial innate immune responses. Lcn2 is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits the in vitro growth of mycobacteria through sequestration of iron uptake. Lcn2-deficient mice are highly susceptible to intratracheal infection with M. tuberculosis. Histological analyses at the early phase of mycobacterial infection in Lcn2-deficient mice reveal increased numbers of mycobacteria in epithelial cell layers, but not in macrophages, in the lungs. Increased intracellular mycobacterial growth is observed in alveolar epithelial cells, but not in alveolar macrophages, from Lcn2-deficient mice. The inhibitory action of Lcn2 is blocked by the addition of endocytosis inhibitors, suggesting that internalization of Lcn2 into the epithelial cells is a prerequisite for the inhibition of intracellular mycobacterial growth. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection, in which Lcn2 mediates anti-mycobacterial innate immune responses within the epithelial cells.