Idan Cohen

IL-1α and IL-1β Recruit Different Myeloid Cells and Promote Different Stages of Sterile Inflammation

The immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current study, we assessed in a comparative manner the role of IL-1α and IL-1β in the initiation and propagation of sterile inflammation induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1α, and not IL-1β, is upregulated and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1α in the initial phase, which correlated with the infiltration of neutrophils, and the expression of IL-1β correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules from cells transfected with either precursor IL-1α or mature IL-1β can recruit neutrophils or macrophages, respectively. Taken together, these data suggest that IL-1α, released from dying cells, initiates sterile inflammation by inducing recruitment of neutrophils, whereas IL-1β promotes the recruitment and retention of macrophages. Overall, our data provide new insight into the biology of IL-1 molecules as well as on the regulation of sterile inflammation.

Differential release of chromatin-bound IL-1α discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation

IL-1α, like IL-1β, possesses multiple inflammatory and immune properties. However, unlike IL-1β, the cytokine is present intracellularly in healthy tissues and is not actively secreted. Rather, IL-1α translocates to the nucleus and participates in transcription. Here we show that intracellular IL-1α is a chromatin-associated cytokine and highly dynamic in the nucleus of living cells. During apoptosis, IL-1α concentrates in dense nuclear foci, which markedly reduces its mobile nature. In apoptotic cells, IL-1α is retained within the chromatin fraction and is not released along with the cytoplasmic contents. To simulate the in vivo inflammatory response to cells undergoing different mechanisms of death, lysates of cells were embedded in Matrigel plugs and implanted into mice. Lysates from cells undergoing necrosis recruited cells of the myeloid lineage into the Matrigel, whereas lysates of necrotic cells lacking IL-1α failed to recruit an infiltrate. In contrast, lysates of cells undergoing apoptotic death were inactive. Cells infiltrating the Matrigel were due to low concentrations (20–50 pg) of the IL-1α precursor containing the receptor interacting C-terminal, whereas the N-terminal propiece containing the nuclear localization site failed to do so. When normal keratinocytes were subjected to hypoxia, the constitutive IL-1α precursor was released into the supernatant. Thus, after an ischemic event, the IL-1α precursor is released by hypoxic cells and incites an inflammatory response by recruiting myeloid cells into the area. Tissues surrounding the necrotic site also sustain damage from the myeloid cells. Nuclear trafficking and differential release during necrosis vs. apoptosis demonstrate that inflammation by IL-1α is tightly controlled.

A Proposed Mechanism for the Inhibitory Effects of Oxidative Stress on Rubisco Assembly and Its Subunit Expression

In Chlamydomonas reinhardtii, a light-induced oxidative stress shifts the glutathione pool toward its oxidized form, resulting in a translational arrest of the large subunit (LSU) of Rubisco. We show here that the translational arrest of LSU is tightly coordinated with cessation of Rubisco assembly, and both processes take place after a threshold level of reactive oxygen species is reached. As a result, the small subunit is also eliminated by rapid degradation. We previously showed that the amino terminus of the LSU could bind RNA in a sequence-independent manner, as it shares a structural similarity with the RNA recognition motif. This domain becomes exposed only under oxidizing conditions, thus restricting the RNA-binding activity. Here we show that in vitro, thiol groups of both subunits become oxidized in the presence of oxidized glutathione. The structural changes are mediated by oxidized glutathione, whereas only very high concentrations of H2O2 confer similar results in vitro. Changes in the redox state of the LSU thiol groups are also observed in vivo, in response to a physiological light shock caused by transfer of cells from low light to high light. We propose that during a photooxidative stress, oxidation of thiol groups occurs already in nascent LSU chains, perhaps hindering their association with chaperones. As a result, their RNA recognition motif domain becomes exposed and will bind any RNA in its vicinity, including its own transcript. Due to this binding the ribosome stalls, preventing the assembly of additional ribosomes on the transcript. Polysome analysis using Suc gradients indeed shows that the rbcL RNA is associated with the polysomal fraction at all times but shifts toward fractions that contain smaller polysomes and monosomes during oxidative stress. Thus, translational arrest of the LSU most likely occurs at a postinitiation stage.

A Deletion Mutation in TMEM38B Associated with Autosomal Recessive Osteogenesis Imperfecta

Autosomal recessive osteogenesis imperfecta (OI) was diagnosed in three unrelated Israeli Bedouin consanguineous families. Fractures were evident in all cases in infancy. Genome‐wide linkage analysis ruled out association with any of the known OI genes, and identified a single homozygosity locus of approximately 2 Mb on chromosome 9 common to all affected individuals (maximum multipoint lod score 6.5). Whole exome sequencing identified only a single mutation within this locus that was shared by all affected individuals: a homozygous deletion mutation of exon 4 of TMEM38B, leading to an early stop codon and a truncated protein, as well as low TMEM38B mRNA levels. TMEM38B encodes TRIC‐B, a ubiquitous component of TRIC, a monovalent cation‐specific channel involved in Ca2+ release from intracellular stores that has been shown to act in cell differentiation. Molecular mechanisms through which a TMEM38B mutation might lead to an OI phenotype are yet to be explored.

Pelizaeus-Merzbacher-like Disease Caused by AIMP1/p43 Homozygous Mutation

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by PLP1 mutations. A similar autosomal-recessive phenotype, Pelizaeus-Merzbacher-like disease (PMLD), has been shown to be caused by homozygous mutations in GJC2 or HSPD1. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD in which linkage to PLP1, GJC2, and HSPD1 was excluded. Through genome-wide homozygosity mapping and mutation analysis, we demonstrated in all affected individuals a homozygous frameshift mutation that fully abrogates the main active domain of AIMP1, encoding ARS-interacting multifunctional protein 1. The mutation fully segregates with the disease-associated phenotype and was not found in 250 Bedouin controls. Our findings are in line with the previously demonstrated inability of mutant mice lacking the AIMP1/p43 ortholog to maintain axon integrity in the central and peripheral neural system.

A Conserved Mechanism Controls Translation of Rubisco Large Subunit in Different Photosynthetic Organisms

We previously proposed a mechanism for control of Rubisco expression and assembly during oxidative stress in Chlamydomonas reinhardtii. The N terminus of the large subunit (LSU) comprises an RNA recognition motif (RRM) that is normally buried in the protein, but becomes exposed under oxidizing conditions when the glutathione pool shifts toward its oxidized form. Thus, de novo translation and assembly of Rubisco LSU stop with similar kinetics and the unpaired small subunit (SSU) is rapidly degraded. Here we show that the structure of the N-terminal domain is highly conserved throughout evolution, despite its relatively low sequence similarity. Furthermore, Rubisco from a broad evolutionary range of photosynthetic organisms binds RNA under oxidizing conditions, with dissociation constant values in the nanomolar range. In line with these observations, oxidative stress indeed causes a translational arrest in land plants as well as in Rhodospirillum rubrum, a purple bacterium that lacks the SSU. We highlight an evolutionary conserved element located within α-helix B, which is located in the center of the RRM and is also involved in the intramolecular interactions between two LSU chains. Thus, assembly masks the N terminus of the LSU hiding the RRM. When assembly is interrupted due to structural changes that occur under oxidizing conditions or in the absence of a dedicated chaperone, the N-terminal domain can become exposed, leading to the translational arrest of Rubisco LSU. Taken together, these results support a model by which LSU translation is governed by its dimerization. In the case that regulation of type I and type II Rubisco is conserved, the SSU does not appear to be directly involved in LSU translation.

Nontransformed, GM-CSF–dependent macrophage lines are a unique model to study tissue macrophage functions

Significance Macrophages—cells crucially involved in defense against infections—exhibit, depending on their anatomical location, distinct biological properties. Studies of the underlying mechanisms are of scientific and clinical interest, but are hampered by the difficulty of obtaining primary tissue macrophages in sufficient numbers and purity. Here, we report the generation of nontransformed murine macrophages, which are similar to alveolar macrophages and can be grown continuously without change of phenotype and in unlimited amounts. Such macrophages helped us to recognize several innate immune properties of alveolar macrophages that are involved in the pathogenesis of infectious lung inflammation. Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF–induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.

Autosomal recessive lethal congenital contractural syndrome type 4 (LCCS4) caused by a mutation in MYBPC1

Autosomal recessive lethal congenital contractural syndrome (LCCS) is a severe form of neuromuscular arthrogryposis. We previously showed that this phenotype is caused in two unrelated inbred Bedouin tribes by different defects in the phosphatidylinositol pathway. However, the molecular basis of the same phenotype in other tribes remained elusive. Whole exome sequencing identified a novel LCCS founder mutation within a minimal shared homozygosity locus of approximately 1 Mb in two affected individuals of different tribes: a homozygous premature stop producing mutation in MYBPC1, encoding myosin‐binding protein C, slow type. A dominant missense mutation in MYBPC1 was previously shown to cause mild distal arthrogryposis. We now show that a recessive mutation abrogating all functional domains in the same gene leads to LCCS. Hum Mutat 33:1435–1438, 2012.

IL-1-induced inflammation promotes development of leishmaniasis in susceptible BALB/c mice

The role of host-derived IL-1 on the course of Leishmania major infection in susceptible BALB/c mice was assessed. Manifestations of the disease were more severe in mice deficient in the physiological inhibitor of IL-1, the IL-1 receptor antagonist (IL-1Ra) in comparison with control mice. In mice lacking one of the IL-1 genes (IL-1alpha or IL-1beta), there was delayed development of the disease and more attenuated systemic inflammatory responses. IL-1alpha-deficient mice were slightly more resistant to L. major infection compared with IL-1beta-knockout mice. During disease progression in IL-1Ra KO and control mice, myeloid-derived suppressor cells invaded the spleen, concomitant to suppression of T cell-mediated immunity and expression of systemic high levels of pro-inflammatory cytokines. In IL-1-deficient mice, T(h)1 responses were still apparent, even at late stages of the disease. Thus, dose-dependent effects of IL-1 were shown to influence the pathogenesis of murine leishamaniasis in susceptible BALB/c mice. Physiological and supra-physiological levels of IL-1 in the microenvironment promoted an exacerbated form of disease, whereas sub-physiological doses of IL-1 induced a less progressive disease. Thus, manipulation of IL-1 levels in the host, using the IL-1Ra or specific antibodies, has the potential to alleviate symptoms of visceral manifestations of leishmaniasis.

Alarmins: Feel the Stress

Over the last decade, danger-associated molecular pattern molecules, or alarmins, have been recognized as signaling mediators of sterile inflammatory responses after trauma and injury. In contrast with the accepted passive release models suggested by the “danger hypothesis,” it was recently shown that alarmins can also directly sense and report damage by signaling to the environment when released from live cells undergoing physiological stress, even without loss of subcellular compartmentalization. In this article, we review the involvement of alarmins such as IL-1α, IL-33, IL-16, and high-mobility group box 1 in cellular and physiological stress, and suggest a novel activity of these molecules as central initiators of sterile inflammation in response to nonlethal stress, a function we denote “stressorins.” We highlight the role of posttranslational modifications of stressorins as key regulators of their activity and propose that targeted inhibition of stressorins or their modifiers could serve as attractive new anti-inflammatory treatments for a broad range of diseases.