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Many bacterial pathogens antagonize host defense responses by translocating effector proteins into cells. It remains an open question how those pathogens not encoding effectors counteract anti-bacterial immunity. Here, we show that Klebsiella pneumoniae exploits the evolutionary conserved innate protein SARM1 to regulate negatively MyD88- and TRIF-governed inflammation, and the activation of the MAP kinases ERK and JNK. SARM1 is required for Klebsiella induction of interleukin-10 (IL-10) by fine-tuning the p38-type I interferon (IFN) axis. SARM1 inhibits the activation of Klebsiella-induced absent in melanoma 2 inflammasome to limit IL-1β production, suppressing further inflammation. Klebsiella exploits type I IFNs to induce SARM1 in a capsule and lipopolysaccharide O-polysaccharide-dependent manner via the TLR4-TRAM-TRIF-IRF3-IFNAR1 pathway. Absence of SARM1 reduces the intracellular survival of K. pneumoniae in macrophages, whereas sarm1-deficient mice control the infection. Altogether, our results illustrate an anti-immunology strategy deployed by a human pathogen. SARM1 inhibition will show a beneficial effect to treat Klebsiella infections.

The challenge of developing gene therapies for genetic forms of blindness is heightened by the heterogeneity of these conditions. However, mechanistic commonalities indicate key pathways that may be targeted in a gene-independent approach. Mitochondrial dysfunction and axon degeneration are common features of many neurodegenerative conditions including retinal degenerations. Here we explore the neuroprotective effect afforded by the absence of sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1), a prodegenerative NADase, in a rotenone-induced mouse model of retinal ganglion cell loss and visual dysfunction. Sarm1 knockout mice retain visual function after rotenone insult, displaying preservation of photopic negative response following rotenone treatment in addition to significantly higher optokinetic response measurements than wild type mice following rotenone. Protection of spatial vision is sustained over time in both sexes and is accompanied by increased RGC survival and additionally preservation of axonal density in optic nerves of Sarm1−/− mice insulted with rotenone. Primary fibroblasts extracted from Sarm1−/− mice demonstrate an increased oxygen consumption rate relative to those from wild type mice, with significantly higher basal, maximal and spare respiratory capacity. Collectively, our data indicate that Sarm1 ablation increases mitochondrial bioenergetics and confers histological and functional protection in vivo in the mouse retina against mitochondrial dysfunction, a hallmark of many neurodegenerative conditions including a variety of ocular disorders.

SUMMARYMany bacterial pathogens antagonize host defence responses by translocating effector proteins into cells. It remains an open question how those pathogens not encoding effectors counteract anti-bacterial immunity. Here, we show thatKlebsiella pneumoniaehijacks the evolutionary conserved innate immune protein SARM1 to control cell intrinsic immunity.Klebsiellaexploits SARM1 to regulate negatively MyD88 and TRIF-governed inflammation, and the activation of the MAP kinases ERK and JNK. SARM1 is required forKlebsiellainduction of IL10 by fine-tuning the p38-type I IFN axis. SARM1 inhibits the activation ofKlebsiella-induced absent in melanoma 2 inflammasome to limit IL1β production, suppressing further inflammation.Klebsiellaexploits type I IFNs to induce SARM1 in a capsule and LPS O-polysaccharide-dependent manner via TLR4-TRAM-TRIF-IRF3-IFNAR1 pathway. Absence of SARM1 reduces the intracellular survival ofK. pneumonaiein macrophages whereassarm1deficient mice control the infection. Altogether, our results illustrate a hitherto unknown anti-immunology strategy deployed by a human pathogen.

SARM1 is an ancient and highly conserved TIR-domain containing protein, with a diverse range of proposed roles in both innate immunity and neuronal death and degeneration. Murine SARM1 has been reported to regulate the transcription of specific chemokines in both neurons and macrophages, however the extent and mechanism by which SARM1 contributes to transcription regulation remains to be fully understood. Here, using RNA sequencing we identify differential gene expression in bone marrow-derived macrophages (BMDM) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared to wild type (WT). However, we show that passenger genes which are derived from the 129 donor strain of mice flank the Sarm1 locus, confounding interpretation of results, since many of the identified differentially regulated genes come from the region containing passenger genes. To re-examine the transcriptional role of SARM1 in the absence of such passenger genes, we generated three different Sarm1-/- mice using CRISPR/Cas9 technology. Vincristine treatment of ex vivo cultured post-natal neurons from these mice confirmed SARM1’s previously identified key function as an executor of axon degeneration. However, using these mice, we show that the absence of SARM1 has no impact on transcription of genes previously shown to be altered in macrophages or in the brainstem. To gain further insight into SARM1 function, we generated and characterized a mouse expressing epitope-tagged SARM1, as it has been difficult to date to confirm which cells and tissues express SARM1 protein. In these mice we see high SARM1 protein expression in the brain and brainstem, and lower but detectable levels in macrophages. Overall, the generation of these next generation SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages but has no general role in transcriptional regulation in these cells, and has provided important new animal models to further explore SARM1 function.

Host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) detect viruses and other pathogens, inducing production of cytokines that cause inflammation and mobilize cells to control infection. Vaccinia virus (VACV) encodes proteins that antagonize these host innate immune responses, and elucidating the mechanisms of action of these viral proteins helped shed light on PRR signaling mechanisms. The VACV virulence factor E3 is one of the most intensely studied VACV proteins and has multiple effects on host cells, many of which cannot be explained by the currently known cellular targets of E3. Here, we report that E3 expression in human monocytes alters TLR2- and TLR8-dependent cytokine induction, and particularly inhibits interleukin (IL)-6. Using MS, we identified DExD/H-box helicase 9 (DHX9) as an E3 target. Although DHX9 has previously been implicated as a PRR for sensing nucleic acid in dendritic cells, we found no role for DHX9 as a nucleic acid-sensing PRR in monocytes. Rather, DHX9 suppression in these cells phenocopied the effects of E3 expression on TLR2- and TLR8-dependent cytokine induction, in that DHX9 was required for all TLR8-dependent cytokines measured, and for TLR2-dependent IL-6. Furthermore, DHX9 also had a cell- and stimulus-independent role in IL-6 promoter induction. DHX9 enhanced NF-κB–dependent IL-6 promoter activation, which was directly antagonized by E3. These results indicate new roles for DHX9 in regulating cytokines in innate immunity and reveal that VACV E3 disrupts innate immune responses by targeting of DHX9.

SARM1 is a toll/interleukin-1 receptor -domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages; however, the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone-marrow-derived macrophages (BMDMs) from C57BL/6 congenic 129 ES cell-derived Sarm1(−/−) mice compared with wild type (WT). However, we found that passenger genes, which are derived from the 129 donor strain of mice that flank the Sarm1 locus, confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1(−/−) mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1's function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To gain further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation and has provided important new models to further explore SARM1 function.

Pattern recognition receptors (PRRs) and inflammasomes are a key part of the anti-viral innate immune system as they detect conserved viral pathogen-associated molecular patterns (PAMPs). A successful host response to viral infections critically depend on the initial activation of PRRs by viruses, mainly by viral DNA and RNA. The signalling pathways activated by PRRs leads to the expression of pro-inflammatory cytokines, to recruit immune cells, and type I and type III interferons which leads to the induction of interferon stimulated genes (ISG), powerful virus restriction factors that establish the "antiviral state". Inflammasomes contribute to anti-viral responses through the maturation of interleukin (IL)-1 and IL-18 and through triggering pyroptotic cell death. The activity of the innate immune system along with the adaptive immune response normally leads to successful virus elimination, although disproportionate innate responses contribute to viral pathology. In this review we will discuss recent insights into the influence of PRR activation and inflammasomes on viral infections and what this means for the mammalian host. We will also comment on how specific PRRs and inflammasomes may be relevant to how SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, interacts with host innate immunity.

This study reports that activation of SARM1 causes destruction of NAD pools in photoreceptor cells of the retina and identifies a role for SARM1-dependent photoreceptor cell death in retinal degeneration., Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of rho−/−sarm1−/− double knockout mice, we demonstrate that genetic deletion of SARM1 promotes both rod and cone photoreceptor cell survival in the rhodopsin knockout (rho−/−) mouse model of photoreceptor degeneration. Finally, we demonstrate that SARM1 deficiency preserves cone visual function in the surviving photoreceptors when assayed by electroretinography. Overall, our data indicate that endogenous SARM1 has the capacity to consume NAD in photoreceptor cells and identifies a previously unappreciated role for SARM1-dependent cell death in photoreceptor cell degeneration.

peer-reviewed There has been a move away from vaccines composed of whole or inactivated antigens toward subunit-based vaccines, which although safe, provide less immunological protection. As a result, the use of adjuvants to enhance and direct adaptive immune responses has become the focus of much targeted bovine vaccine research. However, the mechanisms by which adjuvants work to enhance immunological protection in many cases remains unclear, although this knowledge is critical to the rational design of effective next generation vaccines. This study aimed to investigate the mechanisms by which alum, a commonly used adjuvant in bovine vaccines, enhances IL-1β secretion in bovine peripheral blood mononuclear cells (PBMCs). Unlike the case with human PBMCs, alum promoted IL-1β secretion in a subset of bovine PBMCs without priming with a toll-like receptor agonist. This suggests that PBMCs from some cattle are primed to produce this potent inflammatory cytokine and western blotting confirmed the presence of preexisting pro-IL-1β in PBMCs from a subset of 8-month-old cattle. To address the mechanism underlying alum-induced IL-1β secretion, specific inhibitors identified that alum mediates lysosomal disruption which subsequently activates the assembly of an NLRP3, ASC, caspase-1, and potentially caspase-8 containing complex. These components form an inflammasome, which mediates alum-induced IL-1β secretion in bovine PBMCs. Given the demonstrated role of the NLRP3 inflammasome in regulating adaptive immunity in murine systems, these results will inform further targeted research into the potential of inflammasome activation for rational vaccine design in cattle.

The four Toll/IL-1R domain–containing adaptor proteins MyD88, MAL, TRIF, and TRAM are well established as essential mediators of TLR signaling and gene induction following microbial detection. In contrast, the function of the fifth, most evolutionarily conserved Toll/IL-1R adaptor, sterile α and HEAT/Armadillo motif-containing protein (SARM), has remained more elusive. Recent studies of Sarm−/− mice have highlighted a role for SARM in stress-induced neuronal cell death and immune responses in the CNS. However, whether SARM has a role in immune responses in peripheral myeloid immune cells is less clear. Thus, we characterized TLR-induced cytokine responses in SARM-deficient murine macrophages and discovered a requirement for SARM in CCL5 production, whereas gene induction of TNF, IL-1β, CCL2, and CXCL10 were SARM-independent. SARM was not required for TLR-induced activation of MAPKs or of transcription factors implicated in CCL5 induction, namely NF-κB and IFN regulatory factors, nor for Ccl5 mRNA stability or splicing. However, SARM was critical for the recruitment of transcription factors and of RNA polymerase II to the Ccl5 promoter. Strikingly, the requirement of SARM for CCL5 induction was not restricted to TLR pathways, as it was also apparent in cytosolic RNA and DNA responses. Thus, this study identifies a new role for SARM in CCL5 expression in macrophages.

Assembly of inflammasomes after infection or injury leads to the release of interleukin-1β (IL-1β) and to pyroptosis. After inflammasome activation, cells either pyroptose or enter a hyperactivated state defined by IL-1β secretion without cell death, but what controls these different outcomes is unknown. Here, we show that removal of the Toll-IL-1R protein SARM from macrophages uncouples inflammasome-dependent cytokine release and pyroptosis, whereby cells displayed increased IL-1β production but reduced pyroptosis. Correspondingly, increasing SARM in cells caused less IL-1β release and more pyroptosis. SARM suppressed IL-1β by directly restraining the NLRP3 inflammasome and, hence, caspase-1 activation. Consistent with a role for SARM in pyroptosis, Sarm1-/- mice were protected from lipopolysaccharide (LPS)-stimulated sepsis. Pyroptosis-inducing, but not hyperactivating, NLRP3 stimulants caused SARM-dependent mitochondrial depolarization. Thus, SARM-dependent mitochondrial depolarization distinguishes NLRP3 activators that cause pyroptosis from those that do not, and SARM modulation represents a cell-intrinsic mechanism to regulate cell fate after inflammasome activation.

Summary Toll-like receptors (TLRs) have a central role in innate immunity as they detect conserved pathogen-associated molecular patterns (PAMPs) on a range of microbes, including viruses, leading to innate immune activation and orchestration of the adaptive immune response. To date, a large number of viruses have been shown to trigger innate immunity via TLRs, suggesting that these receptors are likely to be important in the outcome to viral infection. This suggestion is supported by the observation that many viruses have evolved mechanisms not only to evade the innate immune system, but also to subvert it for the benefit of the virus. In this review we will discuss earlier evidence, mainly from knock-out mice studies, implicating TLRs in the innate immune response to viruses, in light of more recent clinical data demonstrating that TLRs are important for anti-viral immunity in humans.

Coupling of M2 and M3 muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M3 receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser789), a putative downstream target of MAP kinases. Alkylation of M3 receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 μM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M2 receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M2receptor activation in smooth muscle.

A key part of the innate immune system is a network of pattern recognition receptors (PRRs) and their associated intracellular signalling pathways. Toll-like receptors (TLRs) are one such group of PRRs that detect pathogen associated molecular patterns (PAMPs). Activation of the TLRs with their respective agonists results in the activation of intracellular signalling pathways leading to the expression of proinflammatory mediators and anti-microbial effector molecules. Activation of the innate immune system through TLRs also triggers the adaptive immune response, resulting in a comprehensive immune program to eradicate invading pathogens. It is now known that immune surveillance and inflammatory responses occur in the central nervous system (CNS). Furthermore it is becoming increasingly clear that TLRs have a role in such CNS responses and are also implicated in the pathogenesis of a number of conditions in the CNS, such as Alzheimer's, stroke and multiple sclerosis. This is likely due to the generation of endogenous TLR agonists in these conditions which amplifies a detrimental neurotoxic inflammatory response. However TLRs in some situations can be neuroprotective, if triggered in a favourable context. This review aims to examine the recent literature on TLRs in the CNS thus demonstrating their importance in a range of infectious and non-infectious diseases of the brain.

Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll-interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction 'downstream' of TRIF but not of MyD88. SARM associated with TRIF, and 'knockdown' of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.

BRCA1 has been implicated in a number of cellular processes, including transcriptional regulation, DNA damage repair, cell cycle arrest, and apoptosis. We identified mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3), an upstream regulator of the c-Jun NH2-terminal kinase/stress-activated protein kinase and p38/MAPK pathways, as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by coimmunoprecipitation in mammalian cells. Deletion mapping demonstrated that amino acids 1611–1863 are required to mediate the interaction with MEKK3 in yeast. BRCA1 disease-associated mutations abrogated the interaction in yeast, and BRCA1 failed to interact with MEKK3 in BRCA1 mutant HCC1937 breast cancer cells. We demonstrate that small interfering RNA-based inhibition of endogenous BRCA1 reduces MEKK3 kinase activity and conversely that inducible expression of BRCA1 activates MEKK3 and p38/MAPK. Finally, we demonstrate using complementary approaches that BRCA1 is required for paclitaxel-induced activation of MEKK3. These data indicate that BRCA1 is a key regulator of the paclitaxel-induced stress response pathway and suggest that the ability of BRCA1 to associate with, and mediate the activation of, MEKK3 represents a potential mechanism through which this pathway is regulated.

We have evaluated the role played by BRCA1 in mediating the phenotypic response to a range of chemotherapeutic agents commonly used in cancer treatment. Here we provide evidence that BRCA1 functions as a differential mediator of chemotherapy-induced apoptosis. Specifically, we demonstrate that BRCA1 mediates sensitivity to apoptosis induced by antimicrotubule agents but conversely induces resistance to DNA-damaging agents. These data are supported by a variety of experimental models including cells with inducible expression of BRCA1, siRNA-mediated inactivation of endogenous BRCA1, and reconstitution of BRCA1-deficient cells with wild-type BRCA1. Most notably we demonstrate that BRCA1 induces a 10-1000-fold increase in resistance to a range of DNA-damaging agents, in particular those that give rise to double-strand breaks such as etoposide or bleomycin. In contrast, BRCA1 induces a1000-fold increase in sensitivity to the spindle poisons, paclitaxel and vinorelbine. Fluorescence-activated cell sorter analysis demonstrated that BRCA1 mediates G(2)/M arrest in response to both antimicrotubule and DNA-damaging agents. However, poly(ADP-ribose) polymerase and caspase-3 cleavage assays indicate that the differential effect mediated by BRCA1 in response to these agents occurs through the inhibition or induction of apoptosis. Therefore, our data suggest that BRCA1 acts as a differential modulator of apoptosis depending on the nature of the cellular insult.

MegaCam is an imaging CCD camera with a 1 square degree field of view for the new MegaPrime prime focus of the 3.6 meter Canada-France-Hawaii Telescope. This CCD camera is fixed on an aluminum structure, called Camembert for its shape, housing a shutter, a filter system and a roll pitch system to tune the CCD mosaic plane. The shutter is made with 1 meter diameter honeycomb half disks that rotates to covers or exposes the CCD mosaic. On this shutter a calibration source is fixed to monitor the CCD and its electronics. The filter system is made of a jukebox with a capacity of eight 30 cm square filters and of a loading arm to place them under the field of view. The instrument was delivered to the CFHT observatory on June 10, 2002 and first light is scheduled in October 2002.

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.