Indiana University-Purdue University Indianapolis (IUPUI) / Store-operated Ca2+ entry (SOCE) is involved in the maintenance of endoplasmic reticulum (ER) Ca2+ levels. The SOCE involves Stromal Interaction Molecule 1 (STIM1), distributed throughout the ER, and Orai1 channels, dispersed on the plasma membrane. SOCE is activated by the depletion of ER Ca2+ causing STIM1 to induce ER expansion and recruits Orai1 channels thus replenishing ER Ca2+. We reported downregulation of STIM1 in human islets from donors with type 2 diabetes (T2D) and in INS-1 β-cells treated with cytokines, and loss of STIM1 expression impairs β-cell SOCE, ER stress, and reduced insulin secretion. However, the regulatory mechanisms of STIM1 downregulation are unknown. To test this, actinomycin D and cycloheximide chase assay was performed to define whether IL-1β treatment impacted STIM1 mRNA or protein half-life. IL-1β had no impact on mRNA or protein decay. MicroRNAs (miRNAs), a class of small non-coding RNAs can regulate gene expression post-transcriptionally by binding to complementary regions in the 3’ untranslated region (UTR) of target mRNAs, affecting mRNA stability and translatability. The objective of this study was to establish miRNA regulation of STIM1 expression and altered SOCE. To identify potential miRNA candidates, RNA sequencing was done in human islets, treated with IL-1β and IFN-γ for 24 hrs. A total of 20 miRNAs were differentially expressed using a FC value of ≥ 1.5 and a p value of < 0.05. Of these, two miRNAs (miR-146a-5p and miR-4640-5p) were predicted by TargetScan to bind the 3’UTR of STIM1.To validate these findings, INS-1 β-cells, and human islets were treated with or without IL-1β. Only miR-146a-5p was upregulated in both systems. Consistent with inverse correlation, INS-1 β-cells transfected with miR-146a-5p mimic showed reduced STIM1 expression. To test whether miR-146a-5p inhibition preserves STIM1 expression, INS1 cells were treated with miR-146a-5p inhibitor along with IL-1β and inhibition of miR-146a-5p led to partial preservation of STIM1 expression. Future studies will test the effect of miR-146a-5p mimics and inhibitors on SOCE. The results indicate that the stress induced by IL-1β leads to induction of miR-146a-5p, which may then target STIM1 mRNA. Such studies could enable broader implementation of miRNA in βcell dysfunction.

Les microARNs (miARN) sont de petits ARN régulant l’expression des gènes au niveau post-transcriptionnel. Ils sont impliqués dans la régulation de nombreux processus biologiques nécessaires au fonctionnement cellulaire, comme le contrôle de la réponse à l’inflammation. Dans ce travail, nous avons évalué l’implication des miARN dans la réponse tubulaire rénale à l’inflammation. Au cours d’un premier travail nous avons étudié par une approche globale le profil d’expression des miARN dans des cellules tubulaires proximales de la lignée HKJ2 exposées à des cytokines pro-inflammatoires. Nous avons ainsi identifié la forte induction de miR-146a en réponse au stimulus inflammatoire. Nous avons ensuite mis en évidence in vitro que l’induction de miR-146a par l’IL-1β dans les cellules HK-2 est secondaire à l’activation de la voie NF-κB, constitue un rétrocontrôle négatif de cette voie et régule l’expression de CXCL8 en aval. In vivo, l’étude du phénotype des souris invalidées pour miR-146a dans un modèle d’agression tubulaire rénale où l’inflammation joue un rôle significatif a mis en évidence une aggravation des lésions tubulaires, de l’infiltrat inflammatoire et de la fibrose interstitielle en réponse à l’ischémie-reperfusion. Le blocage de la signalisation induite par CXCL8 par la réparixine, un inhibiteur du récepteur de CXCL8 (CXCR1), permet de limiter le développement des lésions induites par l’ischémie-reperfusion chez les souris miR-146a-/-. Dans un second travail nous avons exploré le phénotype rénal des souris invalidées pour miR-146a, connues pour développer une auto-immunité. / Independently of its cause, acute kidney injury leads to the development of tubular injury and interstitial inflammation that need to be controlled to avoid fibrosis development. We hypothesized that microRNAs (miRNAs) are involved in the regulation of the balance between lesions and adaptive repair. Using HK2 human proximal tubular epithelial cells, we studied in vitro the response to pro-inflammatory cytokines and the regulation of miR-146a. We explored its targets in HK2 cells after stimulation by IL-1β. In vivo we explored the effect of unilateral renal ischemia-reperfusion injury (IRI) in wild-type or miR-146a invalidated mice. In pro-inflammatory conditions, we identified miR-146a to be transcriptionally upregulated by ligands of the interleukin-1-toll-like receptor signaling in HK2 cells. IL-1β treatment induced miR-146a expression in a time- and concentration-dependent manner through the activation of NF-κB, as confirmed by siRNA and luciferase reporter vector experiments. MiR-146a acted as a negative feedback regulator of this critical pathway by targeting IRAK1, thus decreasing CXCL8/CXCL1 expression by injured tubular cells. In vivo, miR-146a was found to be induced in response to renal IRI in a mouse model of renal unilateral IRI seven days after the injury. In human, miR-146a was found to be induced in the renal allograft of patients who experienced acute tubular necrosis early after transplantation as compared to patients with normal allograft biopsy results (P<0.05). Mir-146a levels were also increased in urine samples collected ten days after renal transplantation in recipients of a deceased donor kidney as compared to recipients of a living donor kidney (P<0.01). In situ hybridization localized up-regulated miR-146a mostly in tubular cells after IRI. Fourteen days after unilateral IRI, miR-146a-/- mice had greater tubular injury, inflammatory infiltrate and fibrosis compared with wild-type mice. Inhibition of the CXCL8/CXCL1 signaling using reparixin, a CXCR2 inhibitor, prevented the development of tubular injury, inflammation and fibrosis after IRI in miR-146a-/- mice. In conclusion, these results highlight miR-146a as a key mediator of the renal response to injury by limiting the consequences of inflammation, a key process in the development of acute and chronic kidney diseases.

Mast cells are resident immune cells abundantly found in the tissue at the host-environment interface, where they play a critical role in inflammatory allergic responses. Mast cell responses may be regulated by the cytokine milieu at the site of inflammation. Recent studies have revealed microRNAs to be important in altering cytokine signaling in immune cells. Here, we demonstrate for the first time that IL-10 and IL-33 induce miR-155 and miR-146a, respectively, to alter mast cell functions. We report that IL-10 enhanced IgE induced activation of mast cells. IL-10 effects are dependent on Stat3 activation, which elicits miR-155 expression, resulting in a loss of suppressor of cytokine signaling-1 (SOCS-1). The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155–deficient mice. Additionally, we show that IL-33 treatment greatly enhances miR-146a expression in mast cells and in mast cell derived exosomes. miR-146a induction is dependent on MyD88 and NFκB and seems to negatively regulate ST2 signaling, which is demonstrated by the hyperresponsiveness of miR-146a knockout BMMC in response to IL-33. Our preliminary data suggest that miR-146a serves as a feedback negative regulator of IL-33 signaling by targeting IRAK proteins. miR-155 and miR-146a are key microRNAs that regulate a range of immune functions. Taken together, our results reveal two novel microRNA pathways that regulate mast cell IgE and IL-33 induced responses.

Mast cells are tissue-resident immune cells known as effector cells for the innate and adaptive immune systems. Mast cells contribute to host defenses against parasites such as large roundworm parasites, bacterial pathogens, and toxins, and participate in wound healing, but they are mostly known for their role in allergic diseases. It has been well established that during allergic diseases, mast cells are stimulated by IgE cross-linkage to release proinflammatory mediators. However, a newly discovered cytokine, IL-33 has also been implicated in allergic disease. Recently, IL-33 has been implicated as a driver of several Type I sensitivities and previous studies have shown that IL-33 can stimulate mast cells in atopic inflammation. Although the importance of IL-33 has been established, there are still several things unknown about IL-33 signaling regulation or treatment. This dissertation will present two separate studies involving the modulation of IL-33-mediated mast cells function In the first study, the effects of fluvastatin are explored. In a previous study, fluvastatin was shown to inhibit proinflammatory functions of IgE crosslinked mast cells. Contrasting to IgE stimulation, fluvastatin augments IL-6 and TNF production in IL-33 stimulated mast cells, but suppressed MCP-1. This phenomenon was seen in mouse and human mast cells in vitro and replicated in a mast cell-dependent murine model of IL-33-induced inflammation in vivo. In the second study, IL-33 was found to induce miR-146a expression in mouse mast cells and mast cell-derived exosomes in vitro, and in plasma exosomes in vivo. IL-33 induced miR-146a was of interest because miR-146a is a known negative regulator of TLR signaling, which shares the MyD88 signaling pathway with IL-33. We found that miR-146a KO mast cells are hyperresponsive to IL-33 stimulation, data that were replicated by suppressing miR-146a-5p in WT mast cells. In an acute mast cell repopulation model, kitW-sh/W-sh mice containing miR-146a KO BMMC had increased IL-33 induced neutrophilia in comparison to their controls. Collectively, these data reveal new IL-33 signaling pathways and means of altering its inflammatory effects on mast cells. Because IL-33 has important roles in allergy and other Th2-mediated diseases, these results advance clinically relevant areas of immunology.

Systemic lupus erythematosus (SLE) is a chronic and complex autoimmune disorder characterized by a failure in the mechanism of self-tolerance and production of autoantibodies, potentially affecting any organ in the body. The genetic factors behind the disease have been extensively studied in the past years and to date a list of more than 30 loci have been associated with SLE. However, very little is known about the functional significance of the risk variants. In this thesis, we focused on the analysis of SLE-associated variants in three genes: interferon regulatory factor 5 (IRF5), CD226 and the microRNA 146a. In paper I, we analyzed four polymorphisms in the IRF5 gene in a large set of individuals from different populations. We replicated a strong association of a promoter indel in our meta-analysis, but expression analysis indicated that it is rather another variant, SNP rs10954213 in the poly(A) signal of the gene that is in fact the major contributor to the altered gene expression in leukocytes. In manuscript II, we further characterized the regulation of IRF5 expression, showing that this gene can be up-regulated by estrogen in PBMCs and monocytes, regardless of the genotype, which could to some extent, explain the sex-bias of SLE. In paper III, we investigated the association of CD226 with SLE and the potential functional effect of the associated variants. The genetic analysis showed an association of a three-SNP-haplotype located at the 3’UTR region of the gene. The risk haplotype correlated with lower CD226 protein expression on the surface of cytotoxic and helper T cells, as well as in NK T cells. Reporter assays pointed to rs727088 in the 3’UTR as the main responsible variant for altered gene expression. In paper IV, we described the association of a variant in microRNA miR-146a, involved in the interferon pathway, with SLE in Europeans, which could in addition be correlated with decreased expression of both mature and primary miR-146a in leukocytes. In summary, we have investigated the genetic association of three genes with SLE in a large cohort of individuals and identified variants responsible for functional alterations of these genes, providing further insight into the pathogenesis of SLE.

The present COVID-19 pandemic has revealed that several characteristics render patients especially prone to developing severe COVID-19 disease, i.e., the male sex, obesity, and old age. An explanation for the observed pattern of vulnerability has been proposed which is based on the concept of low sensitivity of the TLR7-signaling pathway at the time of infection as a common denominator of vulnerable patient groups. We will discuss whether the concept of established TLR-tolerance in macrophages and dendritic cells of the obese and elderly prior to infection can explain not only the vulnerability of these two demographic groups towards development of a severe infection with SARS-CoV-2, but also the observed cytokine response in these vulnerable patients, which is skewed towards pro-inflammatory cytokines with a missing interferon signature.

La sénescence se caractérise par un arrêt en phase G1/S du cycle cellulaire et peut être induit par une variété de stress tels que des télomères trop courts, l’activation d’oncogène ou encore à cause de stress oxydatifs. Cette réponse cellulaire s’accompagne de profonds changements au niveau de l’expression génique et les ARN non codants sont d’importants acteurs de ceux-ci. Bien que cette catégorie d’ARN ait longtemps été considérée comme un sous-produit non fonctionnel de la transcription, on sait maintenant qu’ils sont impliqués dans une pléthore de fonctions essentielles à l’homéostasie de la cellule. Les microARN (miR), de petits ARN non codants d’une vingtaine de nucléotides, sont souvent diminués ou surexprimés dans les maladies, soulignant leurs rôles importants dans le développement de celles-ci. C’est le cas notamment de deux oncomirs, miR-19 et miR-155, qui s’accumulent de manière aberrante dans les cancers hématopoïétiques. En condition normale, STAT5A, qui est souvent dérégulé dans ces cancers, induit SOCS1 qui agit comme un frein sur cette voie de signalisation afin de prévenir une prolifération incontrôlée. Des travaux conduits dans notre laboratoire montrent que SOCS1 est aussi impliqué dans la sénescence, car il est capable d’activer p53, un important suppresseur tumoral. SOCS1 peut être ciblé par les deux oncomirs et nos résultats montrent qu’une inhibition de ces derniers à l’aide d’éponges artificielles favorisait l’accumulation d’un p53 actif. De plus, en intégrant le ribozyme à tête de marteau dans la conception des éponges, nous avons créé une nouvelle génération d’outils (éponges catalytiques) qui sont plus efficaces. Effectivement, l’utilisation de ces éponges contre miR-155 résultait en une diminution de la prolifération, de formation de colonie ainsi que de la migration de cellule de myélome multiple. En second lieu, nous nous sommes penchés sur l’étude d’éponges naturelles dans le contexte de la sénescence. Il existe en effet quelques exemples de lARNnc (Long Non-Coding RNA) qui peuvent agir de la sorte pour un miR donné. Nous pensons que c’est par ce mécanisme de régulation que miR-146a, un miR impliqué dans la réponse anti-inflammatoire, peut s’accumuler dans la sénescence induite par RAS sans toutefois sembler être pleinement actif. Effectivement, les cellules sénescentes sécrètent une variété de facteurs pro-inflammatoires. À l’aide d’une nouvelle technique nommée miR-CLIP, nous avons pu étudier l’interactome de miR-146a et avons identifié plusieurs lARNnc qui selon des outils de prédiction, semblent s’hybrider de manière extensive en région 3’ du miR. Ceci est requis pour l’initiation d’un TDMD (Target-Directed miR Degradation) et nous avons donc investigué la possibilité d’un tel évènement dans la régulation de miR-146a. Nos résultats montrent que la surexpression de XXBAC-B444P24.13 mène à une diminution des niveaux de miR-146a qui n’est pas due à une baisse de sa transcription. Bien que le premier article illustre les avantages d’une éponge catalytique artificielle, le second article suggère que cette stratégie pourrait déjà être en place dans les systèmes biologiques, et ce, de manière naturelle. En effet, une fois miR-146a lié à XXBAC-B444P24.13, ce dernier induirait la dégradation du miR par un TDMD. Ceci ouvre donc la porte au développement d’outils qui pourraient être plus performants à des niveaux d’expression plus bas. / Cellular senescence is characterized by a cell cycle arrest in the G1/S phase and can be induced by a variety of stresses which include telomere shortening, oncogene activation or oxidative stress. Its establishment is known to require changes in the genetic expression program and non-coding RNA play an important part in this phenomenon. For a long time, this RNA subtype was considered to only be a transcriptional byproduct, but we now know that they are involved in a plethora of functions which are essential to cell homeostasis. Various diseases display aberrant expression of microRNA (miR), small non-coding RNA of 18-22 nucleotides, suggesting they are involved in their development. Such is the case for miR-19 and miR-155, two oncomirs which are found to be overexpressed in hematopoietic cancers. In normal conditions, STAT5A, which is often found dysregulated in those cancers, induces SOCS1 which acts as a retro-inhibitor of this signaling pathway, preventing uncontrolled proliferation. Furthermore, our lab has shown that SOCS1 can also be involved in senescence by facilitating p53 activation. SOCS1 can be targeted by both oncomirs and our results show that artificial sponges, that inhibit miR-19 or miR-155’s functions, lead to the activation of p53. Also, we have incorporated the hammerhead ribozyme in the miR binding sites in the sponge, creating a sponge 2.0 (catalytic sponges). Expressing the latter in a multiple myeloma cell line (RPMI8226) resulted in less proliferation, colony formation and migration. Secondly, we aimed at studying natural sponges in the context of senescence. Indeed, there are quite a few examples of lncRNA (Long Non-Coding RNA) acting as a miRNA inhibitor by quenching them. We think that this mode of regulation could provide an explanation as to how an anti-inflammatory miR, miR-146a, can accumulate in senescence even though it is a pro-inflammatory response. Using a novel technique called miR-CLIP, we were able to study specifically miR-146a’s interactome and have found that it can interact with many lncRNAs. Interestingly, using computational tools, we noticed that miR-146a was predicted to interact with extensive 3’ end hybridization with a number of these lncRNA. This characteristic is known to be required to induce TDMD (Target-Directed miR Degradation). Indeed, when we overexpressed XXBAC-B444P24.13, miR-146a levels went down and this is not caused by a decrease in transcription of the miR. In the first part of this thesis, we show that artificial catalytic sponges have an advantage over a more “classical” design. This is further supported by the fact that this strategy seems to be employed in nature. Indeed, we might have uncovered a lncRNA that when bound to by miR-146a would lead to its degradation using TDMD. This could be taken advantage of in the development of new tools for miR inhibition that would be more powerful and could be potentially used at lower levels of expression.

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