Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation.

Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although our knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. Here, we identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.

Gene expression profiling identifies the zinc-finger protein Charlatan as a regulator of intestinal stem cells in Drosophila.

Intestinal stem cells (ISCs) in the adult Drosophila midgut can respond to tissue damage and support repair. We used genetic manipulation to increase the number of ISC-like cells in the adult midgut and performed gene expression profiling to identify potential ISC regulators. A detailed analysis of one of these potential regulators, the zinc-finger protein Charlatan, was carried out. MARCM clonal analysis and RNAi in precursor cells showed that loss of Chn function caused severe ISC division defects, including loss of EdU incorporation, phosphorylated histone 3 staining and expression of the mitotic protein Cdc2. Loss of Charlatan also led to a much reduced histone acetylation staining in precursor cells. Both the histone acetylation and ISC division defects could be rescued by the simultaneous decrease of the Histone Deacetylase 2. The overexpression of Charlatan blocked differentiation reversibly, but loss of Charlatan did not lead to automatic differentiation. The results together suggest that Charlatan does not simply act as an anti-differentiation factor but instead functions to maintain a chromatin structure that is compatible with stem cell properties, including proliferation.

Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila.

Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. Here, we show that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, we define a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP.

Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants.

Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.

Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine.

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc+ in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut.

We reveal surprising similarities between homeostatic cell turnover in adult Drosophila midguts and "undead" apoptosis-induced compensatory proliferation (AiP) in imaginal discs. During undead AiP, immortalized cells signal for AiP, allowing its analysis. Critical for undead AiP is the Myo1D-dependent localization of the initiator caspase Dronc to the plasma membrane. Here, we show that Myo1D functions in mature enterocytes (ECs) to control mitotic activity of intestinal stem cells (ISCs). In Myo1D mutant midguts, many signaling events involved in AiP (ROS generation, hemocyte recruitment, and JNK signaling) are affected. Importantly, similar to AiP, Myo1D is required for membrane localization of Dronc in ECs. We propose that ECs destined to die transiently enter an undead-like state through Myo1D-dependent membrane localization of Dronc, which enables them to generate signals for ISC activity and their replacement. The concept of transiently "undead" cells may be relevant for other stem cell models in flies and mammals.

IgA MAb blocks SARS-CoV-2 Spike-ACE2 interaction providing mucosal immunity.

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity or as a therapeutic has yet been developed to SARS-CoV-2. In this study we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks hACE2 receptor binding, by completely overlapping the hACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in human epithelial cells expressing hACE2. SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.

A cross-reactive human IgA monoclonal antibody blocks SARS-CoV-2 spike-ACE2 interaction.

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.

Oncogenic Pathways and Loss of the Rab11 GTPase Synergize To Alter Metabolism in Drosophila.

Colorectal cancer is a complex disease driven by well-established mutations such as APC and other yet to be identified pathways. The GTPase Rab11 regulates endosomal protein trafficking, and previously we showed that loss of Rab11 caused intestinal inflammation and hyperplasia in mice and flies. To test the idea that loss of Rab11 may promote cancer progression, we have analyzed archival human patient tissues and observed that 51 out of 70 colon cancer tissues had lower Rab11 protein staining. By using the Drosophila midgut model, we have found that loss of Rab11 can lead to three changes that may relate to cancer progression. First is the disruption of enterocyte polarity based on staining of the FERM domain protein Coracle. Second is an increased proliferation due to an increased expression of the JAK-STAT pathway ligand Upd3. Third is an increased expression of ImpL2, which is an IGFBP7 homolog and can suppress metabolism. Furthermore, loss of Rab11 can act synergistically with the oncoprotein RasV12 to regulate these cancer-related phenotypes.

Plasma Membrane Localization of Apoptotic Caspases for Non-apoptotic Functions.

Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. Here, we report that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. We propose that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib RasV12 model. Together, we identified a new function of Myo1D for AiP and tumorigenesis, and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane.

Toll-like receptor 9 ligand blocks osteoclast differentiation through induction of phosphatase.

UNLABELLED: CpG-ODN, in addition to stimulation of osteoclastogenic signals in early osteoclast precursors, also induces phosphatase, shifting the pattern of ERK phosphorylation from sustained to transient. This shift results in the degradation of c-fos, an essential molecule for osteoclast differentiation. Therefore, CpG-ODN blocks osteoclast differentiation. INTRODUCTION: Activation of either Toll-like receptor 9 (TLR9) or RANK induces similar responses in osteoclast precursors. Paradoxically, activation of TLR9 results in inhibition of RANKL-induced osteoclastogenesis. MATERIALS AND METHODS: We used bone marrow-derived osteoclast precursors. Analyses of signaling molecules phosphorylation were performed using Western blotting. Different levels of gene expression analyses were performed using RT-PCR, Northern, and run-on analyses (for RNA), and EMSA, Western, and pulse-chase experiments (for protein). Phosphatase activity was measured spectrophotometrically. RESULTS: We found that RANKL and TLR9 ligand, oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN), induce sustained and transient extracellular signal-regulated kinase (ERK) phosphorylation, respectively. Furthermore, together they induce a transient phosphorylation of ERK. The duration of ERK phosphorylation is a key factor in determining induction of c-fos, a protein critical for osteoclastogenesis. Indeed, we found that CpG-ODN does not induce c-fos and inhibits its induction by RANKL by enhancing c-fos mRNA and protein degradation. Our observation that CpG-ODN, but not RANKL, induces the expression of the phosphatase PP2A suggests that CpG-ODN exerts its inhibitory activity by induction of ERK dephosphorylation. Moreover, together with the phosphatase inhibitor okadaic acid, CpG-ODN induces sustained ERK phosphorylation and c-fos expression. CONCLUSIONS: Our findings suggest that the increased rate of c-fos degradation by the TLR9 ligand mediates the inhibition of RANKL-induced osteoclast differentiation. The TLR9 ligand, through induction of dephosphorylation, prevents the sustained ERK phosphorylation needed for maintaining high c-fos levels that are essential for osteoclast differentiation.

Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages.

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ("undead") model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer.

Differential contribution of osteoclast- and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis.

UNLABELLED: CpG-ODNs modulate osteoclast differentiation through Toll-like receptor 9 (TLR9). Using TLR9-deficient mice, we found that activation of TLR9 on both osteoclast precursors and osteoblasts mediate the osteoclastogenic effect of CpG-ODN. Osteoclastic TLR9 is more important for this activity. INTRODUCTION: Bacterial infections cause pathological bone loss by accelerating differentiation and activation of the osteoclast. A variety of bacteria-derived molecules have been shown to enhance osteoclast differentiation through activation of Toll-like receptors (TLRs). We have shown that CpG-oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA and exerting their cellular activities through TLR9, modulate osteoclast differentiation in a complex manner: the ODNs inhibit the activity of the physiological osteoclast differentiation factor RANKL in early osteoclast precursors (OCPs) but markedly stimulate osteoclastogenesis in cells primed by RANKL. MATERIALS AND METHODS: Osteoclast precursors and osteoblasts from TLR9-deficient (TLR9-/-) and wildtype (TLR9+/+) mice were used for in vitro analyses of osteoclast differentiation and modulation of signal transduction and gene expression. RESULTS: As expected CpG-ODN did not exert any activity in cells derived from TLR9-/-mice; these cells, however, responded in a normal manner to other stimuli. Using bone marrow/osteoblasts co-cultures from all possible combinations of TLR9-/- and TLR9+/+ mice-derived cells, we showed that TLR9 in the two lineages is required for CpG-ODN induction of osteoclastogenesis. CONCLUSIONS: CpG-ODN modulates osteoclastogenesis in a TLR9-dependent manner. Activation of TLR9 in bone marrow-derived osteoclasts precursors is more crucial to induction of osteoclastogenesis than activation of the osteoblastic TLR9.

Bunched and Madm Function Downstream of Tuberous Sclerosis Complex to Regulate the Growth of Intestinal Stem Cells in Drosophila.

The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. We show here that the leucine zipper protein Bunched and the adaptor protein Madm are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, our data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells.

Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila.

Intestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine cells, in regulating intestinal stem cells remains unclear. We show here that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth.

The conserved misshapen-warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila.

Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. Here, we show that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP.

Be a good neighbor: organ-to-organ communication during the innate immune response.

Local infection in the Drosophila larval intestine elicits a systemic immune reaction in fat bodies. In this issue, Wu and colleagues (2012) show that this is a reactive oxygen species-dependent communication.

Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells.

Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. We demonstrate in this paper that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, we report in this paper that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs expressed proper stem cell markers, did not differentiate, and had defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc was sufficient to rescue the division defect. The TSC mutant guts had a thinner epithelial structure than wild-type tissues, and the mutant flies were more susceptible to tissue damage. Therefore, we have uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division.

Tissue damage-induced intestinal stem cell division in Drosophila.

Stem cell division is essential for tissue integrity during growth, aging, and pathogenic assaults. Adult gastrointestinal tract encounters numerous stimulations, and impaired tissue regeneration may lead to inflammatory diseases and cancer. Intestinal stem cells in adult Drosophila have recently been identified and shown to replenish the various cell types within the midgut. However, it is not known whether these intestinal stem cells can respond to environmental challenges. By feeding dextran sulfate sodium and bleomycin to flies and by expressing apoptotic proteins, we show that Drosophila intestinal stem cells can increase the rate of division in response to tissue damage. Moreover, if tissue damage results in epithelial cell loss, the newly formed enteroblasts can differentiate into mature epithelial cells. By using this newly established system of intestinal stem cell proliferation and tissue regeneration, we find that the insulin receptor signaling pathway is required for intestinal stem cell division.