TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

Acute systemic inflammation critically alters the function of the immune system, often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus, systemic inflammation results in acute thymic atrophy and, consequently, impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here, we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy, while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs), while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs), where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture, indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis, TL1A, and IL-18 synergistically increased GM-CSF levels in the NTOC, which was mainly produced by thymic ILC1s. In support, TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade, revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together, our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while  promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner.

Amoxicillin treatment of pneumococcal pneumonia impacts bone marrow neutrophil maturation and function.

Pneumonia caused by Streptococcus pneumoniae is a leading cause of death worldwide. A growing body of evidence indicates that the successful treatment of bacterial infections results from synergy between antibiotic-mediated direct antibacterial activity and the host's immune defenses. However, the mechanisms underlying the protective immune responses induced by amoxicillin, a ß-lactam antibiotic used as the first-line treatment of S. pneumoniae infections, have not been characterized. A better understanding of amoxicillin's effects on host-pathogen interactions might facilitate the development of other treatment options. Given the crucial role of neutrophils in the control of S. pneumoniae infections, we decided to investigate amoxicillin's impact on neutrophil development in a mouse model of pneumococcal superinfection. A single therapeutic dose of amoxicillin almost completely eradicated the bacteria and prevented local and systemic inflammatory responses. Interestingly, in this context, amoxicillin treatment did not impair the emergency granulopoiesis triggered in the bone marrow by S. pneumoniae. Importantly, treatment of pneumonia with amoxicillin was associated with a greater mature neutrophil count in the bone marrow; these neutrophils had specific transcriptomic and proteomic profiles. Furthermore, amoxicillin-conditioned, mature neutrophils in the bone marrow had a less activated phenotype and might be rapidly mobilized in peripheral tissues in response to systemic inflammation. Thus, by revealing a novel effect of amoxicillin on the development and functions of bone marrow neutrophils during S. pneumoniae pneumonia, our findings provide new insights into the impact of amoxicillin treatment on host immune responses.

The RNA m6A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression.

Emergency granulopoiesis and neutrophil mobilization that can be triggered by granulocyte colony-stimulating factor (G-CSF) through its receptor G-CSFR are essential for antibacterial innate defense. However, the epigenetic modifiers crucial for intrinsically regulating G-CSFR expression and the antibacterial response of neutrophils remain largely unclear. N6-methyladenosine (m6A) RNA modification and the related demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, but their roles in neutrophil production and mobilization are still unknown. We used cecal ligation and puncture (CLP)-induced polymicrobial sepsis to model systemic bacterial infection, and we report that ALKBH5 is required for emergency granulopoiesis and neutrophil mobilization. ALKBH5 depletion significantly impaired the production of immature neutrophils in the bone marrow of septic mice. In addition, Alkbh5-deficient septic mice exhibited higher retention of mature neutrophils in the bone marrow and defective neutrophil release into the circulation, which led to fewer neutrophils at the infection site than in their wild-type littermates. During bacterial infection, ALKBH5 imprinted production- and mobilization-promoting transcriptome signatures in both mouse and human neutrophils. Mechanistically, ALKBH5 erased m6A methylation on the CSF3R mRNA to increase the mRNA stability and protein expression of G-CSFR, consequently upregulating cell surface G-CSFR expression and downstream STAT3 signaling in neutrophils. The RIP-qPCR results confirmed the direct binding of ALKBH5 to the CSF3R mRNA, and the binding strength declined upon bacterial infection, accounting for the decrease in G-CSFR expression on bacteria-infected neutrophils. Considering these results collectively, we define a new role of ALKBH5 in intrinsically driving neutrophil production and mobilization through m6A demethylation-dependent posttranscriptional regulation, indicating that m6A RNA modification in neutrophils is a potential target for treating bacterial infections and neutropenia.

Hematopoietic stem cells undergo a lymphoid to myeloid switch in early stages of emergency granulopoiesis.

Emergency granulopoiesis is the enhanced and accelerated production of granulocytes that occurs during acute infection. The contribution of hematopoietic stem cells (HSCs) to this process was reported; however, how HSCs participate in emergency granulopoiesis remains elusive. Here, using a mouse model of emergency granulopoiesis we observe transcriptional changes in HSCs as early as 4 h after lipopolysaccharide (LPS) administration. We observe that the HSC identity is changed towards a myeloid-biased HSC and show that CD201 is enriched in lymphoid-biased HSCs. While CD201 expression under steady-state conditions reveals a lymphoid bias, under emergency granulopoiesis loss of CD201 marks the lymphoid-to-myeloid transcriptional switch. Mechanistically, we determine that lymphoid-biased CD201+ HSCs act as a first response during emergency granulopoiesis due to direct sensing of LPS by TLR4 and downstream activation of NF-κΒ signaling. The myeloid-biased CD201- HSC population responds indirectly during an acute infection by sensing G-CSF, increasing STAT3 phosphorylation, and upregulating LAP/LAP* C/EBPß isoforms. In conclusion, HSC subpopulations support early phases of emergency granulopoiesis due to their transcriptional rewiring from a lymphoid-biased to myeloid-biased population and thus establishing alternative paths to supply elevated numbers of granulocytes.

Method for Visualization of Emergency Granulopoiesis in the Zebrafish Embryo.

Emergency granulopoiesis (EG) is a response to severe inflammation in which increased neutrophils are generated in the hematopoietic tissue. Photolabeling is utilized to distinguish newly developed neutrophils from existing neutrophils. However, this technique requires a strong laser line and labels subsets of the existing neutrophils. Here we create a transgenic zebrafish line that expresses a time-dependent switch from green fluorescent protein (GFP) to red fluorescent protein (RFP) in neutrophils, which allows quantification of EG using simple GFP/RFP ratiometric imaging.

BCG-Induced Immune Training: Interplay between Trained Immunity and Emergency Granulopoiesis.

Bacille Calmette-Guérin (BCG) is the most commonly administered vaccine in human history. The medical application of BCG extends far beyond the fight against tuberculosis. Despite its stellar medical record over 100 years, insight into how BCG provides this vast range of benefits is largely limited, both for its pathogen-specific (tuberculosis) as well as pathogen-agnostic (other infections, autoimmunity, allergies, and cancer) effects. Trained immunity and emergency granulopoiesis have been identified as mediating BCG's pathogen-agnostic effects, for which some of the molecular mechanisms have been delineated. Upon review of the existing evidence, we postulate that emergency granulopoiesis and trained immunity are a continuum of the same effect cascade. In this context, we highlight that BCG's pathogen-agnostic benefits could be optimized by taking advantage of the age of the recipient and route of BCG administration.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Neutrophil Homeostasis and Emergency Granulopoiesis: The Example of Systemic Juvenile Idiopathic Arthritis.

Neutrophils are key pathogen exterminators of the innate immune system endowed with oxidative and non-oxidative defense mechanisms. More recently, a more complex role for neutrophils as decision shaping cells that instruct other leukocytes to fine-tune innate and adaptive immune responses has come into view. Under homeostatic conditions, neutrophils are short-lived cells that are continuously released from the bone marrow. Their development starts with undifferentiated hematopoietic stem cells that pass through different immature subtypes to eventually become fully equipped, mature neutrophils capable of launching fast and robust immune responses. During severe (systemic) inflammation, there is an increased need for neutrophils. The hematopoietic system rapidly adapts to this increased demand by switching from steady-state blood cell production to emergency granulopoiesis. During emergency granulopoiesis, the de novo production of neutrophils by the bone marrow and at extramedullary sites is augmented, while additional mature neutrophils are rapidly released from the marginated pools. Although neutrophils are indispensable for host protection against microorganisms, excessive activation causes tissue damage in neutrophil-rich diseases. Therefore, tight regulation of neutrophil homeostasis is imperative. In this review, we discuss the kinetics of neutrophil ontogenesis in homeostatic conditions and during emergency myelopoiesis and provide an overview of the different molecular players involved in this regulation. We substantiate this review with the example of an autoinflammatory disease, i.e. systemic juvenile idiopathic arthritis.

No evidence that plasmablasts transdifferentiate into developing neutrophils in severe COVID-19 disease.

OBJECTIVES: A recent single-cell RNA sequencing study by Wilk et al. suggested that plasmablasts can transdifferentiate into 'developing neutrophils' in patients with severe COVID-19 disease. We explore the evidence for this. METHODS: We downloaded the original data and code used by the authors in their study to replicate their findings and explore the possibility that regressing out variables may have led the authors to overfit their data. RESULTS: The lineage relationship between plasmablasts and developing neutrophils breaks down when key features are not regressed out, and the data are not overfitted during the analysis. CONCLUSION: Plasmablasts do not transdifferentiate into developing neutrophils. The single-cell RNA sequencing is a powerful technique for biological discovery and hypothesis generation. However, caution should be exercised in the bioinformatic analysis and interpretation of the data and findings cross-validated by orthogonal techniques.

Neutrophils at the crossroads of acute viral infections and severity.

Neutrophils are versatile immune effector cells essential for mounting a first-line defense against invading pathogens. However, uncontrolled activation can lead to severe life-threatening complications. Neutrophils exist as a heterogeneous population, and their interaction with pathogens and other immune cells may shape the outcome of the host immune response. Diverse classes of viruses, including the recently identified novel SARS-CoV-2, have shown to alter the various aspects of neutrophil biology, offering possibilities for selective intervention. Here, we review heterogeneity within the neutrophil population, highlighting the functional consequences of circulating phenotypes and their critical involvement in exaggerating protective and pathological immune responses against the viruses. We discuss the recent findings of neutrophil extracellular traps (NETs) in COVID-19 pathology and cover other viruses, where neutrophil biology and NETs are crucial for developing disease severity. In the end, we have also pointed out the areas where neutrophil-mediated responses can be finely tuned to outline opportunities for therapeutic manipulation in controlling inflammation against viral infection.

Non-specific effects of BCG vaccination on neutrophil and lymphocyte counts of healthy neonates from a developed country.

BCG vaccination is known to reduce neonatal mortality from infections in a pathogen-agnostic manner. In this observational study we report on whether an emergency granulopoietic response is elicited in term babies from a developed country following BCG vaccination. We studied a cohort of neonates re-admitted to the hospital from home for feeding support separated into 2 groups dependent on whether they had received BCG vaccination. Clinical data including gender, weight, gestational age, method of feeding and full blood count results were retrieved retrospectively. While lymphocyte counts increase following BCG vaccination irrespective of gender and in proportion with the time elapsed after vaccination, the increase in neutrophil counts, is only observed in boys. This increase appears to be temporary. Our results confirm the presence of emergency granulopoiesis following BCG vaccination in a neonatal cohort from a developed country. However, this effect appears to be gender-specific and is present only in boys.

A distinct innate immune signature marks progression from mild to severe COVID-19.

Coronavirus disease 2019 (COVID-19) manifests with a range of severities, but immune signatures of mild and severe disease are still not fully understood. Here, we use mass cytometry and targeted proteomics to profile the innate immune response of patients with mild or severe COVID-19 and of healthy individuals. Sampling at different stages allows us to reconstruct a pseudo-temporal trajectory of the innate response. A surge of CD169+ monocytes associated with an IFN-γ+MCP-2+ signature rapidly follows symptom onset. At later stages, we observe a persistent inflammatory phenotype in patients with severe disease, dominated by high CCL3 and CCL4 abundance correlating with the re-appearance of CD16+ monocytes, whereas the response of mild COVID-19 patients normalizes. Our data provide insights into the dynamic nature of inflammatory responses in COVID-19 patients and identify sustained innate immune responses as a likely mechanism in severe patients, thus supporting the investigation of targeted interventions in severe COVID-19.

Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish.

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

An aberrantly sustained emergency granulopoiesis response accelerates postchemotherapy relapse in MLL1-rearranged acute myeloid leukemia in mice.

Acute myeloid leukemia (AML) with mixed lineage leukemia 1 (MLL1) gene rearrangement is characterized by increased expression of a set of homeodomain transcription factors, including homeobox A9 (HOXA9) and HOXA10. The target genes for these regulators include fibroblast growth factor 2 (FGF2) and Ariadne RBR E3 ubiquitin ligase 2 (ARIH2). FGF2 induces leukemia stem cell expansion in MLL1-rearranged AML. ARIH2 encodes TRIAD1, an E3 ubiquitin ligase required for termination of emergency granulopoiesis and leukemia suppressor function in MLL1-rearranged AML. Receptor tyrosine kinases (RTKs), including the FGF receptor, are TRIAD1 substrates that are possibly relevant to these activities. Using transcriptome analysis, we found increased activity of innate immune response pathways and RTK signaling in bone marrow progenitors from mice with MLL1-rearranged AML. We hypothesized that sustained RTK signaling, because of decreased TRIAD1 activity, impairs termination of emergency granulopoiesis during the innate immune response and contributes to leukemogenesis in this AML subtype. Consistent with this, we found aberrantly sustained emergency granulopoiesis in a murine model of MLL1-rearranged AML, associated with accelerated leukemogenesis. Treating these mice with an inhibitor of TRIAD1-substrate RTKs terminated emergency granulopoiesis, delayed leukemogenesis during emergency granulopoiesis, and normalized innate immune responses when combined with chemotherapy. Emergency granulopoiesis also hastened postchemotherapy relapse in mice with MLL1-rearranged AML, but remission was sustained by ongoing RTK inhibition. Our findings suggest that the physiological stress of infectious challenges may drive AML progression in molecularly defined subsets and identify RTK inhibition as a potential therapeutic approach to counteract this process.

Kidney injury enhances renal G-CSF expression and modulates granulopoiesis and human neutrophil CD177 in vivo.

Kidney injury significantly increases overall mortality. Neutrophilic granulocytes (neutrophils) are the most abundant human blood leukocytes. They are characterized by a high turnover rate, chiefly controlled by granulocyte colony stimulating factor (G-CSF). The role of kidney injury and uremia in regulation of granulopoiesis has not been reported. Kidney transplantation, which inherently causes ischemia-reperfusion injury of the graft, elevated human neutrophil expression of the surface glycoprotein CD177. CD177 is among the most G-CSF-responsive neutrophil genes and reversibly increased on neutrophils of healthy donors who received recombinant G-CSF. In kidney graft recipients, a transient rise in neutrophil CD177 correlated with renal tubular epithelial G-CSF expression. In contrast, CD177 was unaltered in patients with chronic renal impairment and independent of renal replacement therapy. Under controlled conditions of experimental ischemia-reperfusion and unilateral ureteral obstruction injuries in mice, renal G-CSF mRNA and protein expression significantly increased and systemic neutrophilia developed. Human renal tubular epithelial cell G-CSF expression was promoted by hypoxia and proinflammatory cytokine interleukin 17A in vitro. Clinically, recipients of ABO blood group-incompatible kidney grafts developed a larger rise in neutrophil CD177. Their grafts are characterized by complement C4d deposition on the renal endothelium, even in the absence of rejection. Indeed, complement activation, but not hypoxia, induced primary human endothelial cell G-CSF expression. Our data demonstrate that kidney injury induces renal G-CSF expression and modulates granulopoiesis. They delineate differential G-CSF regulation in renal epithelium and endothelium. Altered granulopoiesis may contribute to the systemic impact of kidney injury.

[Differentiation and proliferation of hematopoietic stem cells are regulated by C/EBPß, a transcription factor required for emergency granulopoiesis].

Under stress conditions such as infection, inflammation, and hematopoietic recovery following chemotherapy or transplantation, the hematopoietic system is required to meet the increasing demands, especially from myeloid cells. Therefore, an understanding of the molecular mechanism underlying stress hematopoiesis is clinically imperative. We previously showed that C/EBPß, which is a transcription factor required for emergency granulopoiesis, plays a pivotal role at the level of hematopoietic stem/progenitor cells under stress conditions. Upon exposure to stress, the C/EBPß protein is upregulated in the hematopoietic stem cells. A close examination of C/EBPß knockout mice revealed that C/EBPß regulates the proliferation and differentiation of hematopoietic stem cells at the cost of the self-renewing activity. Further elucidation of the functions and regulation of C/EBPß in hematopoietic stem cells will facilitate an understanding of stress hematopoiesis.

Shigella-Induced Emergency Granulopoiesis Protects Zebrafish Larvae from Secondary Infection.

Emergency granulopoiesis is a hematopoietic program of stem cell-driven neutrophil production used to counteract immune cell exhaustion following infection. Shigella flexneri is a Gram-negative enteroinvasive pathogen controlled by neutrophils. In this study, we use a Shigella-zebrafish (Danio rerio) infection model to investigate emergency granulopoiesis in vivo We show that stem cell-driven neutrophil production occurs in response to Shigella infection and requires macrophage-independent signaling by granulocyte colony-stimulating factor (Gcsf). To test whether emergency granulopoiesis can function beyond homoeostasis to enhance innate immunity, we developed a reinfection assay using zebrafish larvae that have not yet developed an adaptive immune system. Strikingly, larvae primed with a sublethal dose of Shigella are protected against a secondary lethal dose of Shigella in a type III secretion system (T3SS)-dependent manner. Collectively, these results highlight a new role for emergency granulopoiesis in boosting host defense and demonstrate that zebrafish larvae can be a valuable in vivo model to investigate innate immune memory.IMPORTANCEShigella is an important human pathogen of the gut. Emergency granulopoiesis is the enhanced production of neutrophils by hematopoietic stem and progenitor cells (HSPCs) upon infection and is widely considered a homoeostatic mechanism for replacing exhausted leukocytes. In this study, we developed a Shigella-zebrafish infection model to investigate stem cell-driven emergency granulopoiesis. We discovered that zebrafish initiate granulopoiesis in response to Shigella infection, via macrophage-independent signaling of granulocyte colony-stimulating factor (Gcsf). Strikingly, larvae primed with a sublethal dose of Shigella are protected against a secondary lethal dose of Shigella in a type III secretion system (T3SS)-dependent manner. Taken together, we show that zebrafish infection can be used to capture Shigella-mediated stem cell-driven granulopoiesis and provide a new model system to study stem cell biology in vivo Our results also highlight the potential of manipulating stem cell-driven granulopoiesis to boost innate immunity and combat infectious disease.

Use of zebrafish to study Shigella infection.

Shigella is a leading cause of dysentery worldwide, responsible for up to 165 million cases of shigellosis each year. Shigella is also recognised as an exceptional model pathogen to study key issues in cell biology and innate immunity. Several infection models have been useful to explore Shigella biology; however, we still lack information regarding the events taking place during the Shigella infection process in vivo Here, we discuss a selection of mechanistic insights recently gained from studying Shigella infection of zebrafish (Danio rerio), with a focus on cytoskeleton rearrangements and cellular immunity. We also discuss how infection of zebrafish can be used to investigate new concepts underlying infection control, including emergency granulopoiesis and the use of predatory bacteria to combat antimicrobial resistance. Collectively, these insights illustrate how Shigella infection of zebrafish can provide fundamental advances in our understanding of bacterial pathogenesis and vertebrate host defence. This information should also provide vital clues for the discovery of new therapeutic strategies against infectious disease in humans.

Transcriptional Regulation of Emergency Granulopoiesis in Leukemia.

Neutropenic conditions are prevalent in leukemia patients and are often associated with increased susceptibility to infections. In fact, emergency granulopoiesis (EG), a process regulating neutrophil homeostasis in inflammatory conditions and infections, may occur improperly in leukemic conditions, leading to reduced neutrophil counts. Unfortunately, the mechanisms central to dysfunctional EG remain understudied in both leukemia patients and leukemic mouse models. However, despite no direct studies on EG response in leukemia are reported, recently certain transcription factors (TFs) have been found to function at the crossroads of leukemia and EG. In this review, we present an update on TFs that can potentially govern the fate of EG in leukemia. Transcriptional control of Fanconi DNA repair pathway genes is also highlighted, as well as the newly discovered role of Fanconi proteins in innate immune response and EG. Identifying the TFs regulating EG in leukemia and dissecting their underlying mechanisms may facilitate the discovery of therapeutic drugs for the treatment of neutropenia.