Effect of Decreased ERK/MAPK Signaling on Autoimmunity and Disease

Decreased ERK/MAPK signaling leads to autoimmunity and diseases

INTRODUCTION

Mitogen-activated protein kinase (MAPK) signaling pathways consist of cascades of protein kinases that link extracellular stimuli to targets located in different cellular compartments¹. In mammals, the extracellular signal-regulated kinase (ERK) pathway, constitute the best-characterized family of MAPKs. The ERK pathway is activated by multiple surface receptors including growth factor receptors, B and T cell antigen receptors and G protein coupled receptor leading to elicit various physiological outputs including fate determination, differentiation, proliferation, survival, migration and apoptosis^{1,2,3,4,5,6,7,8}. Following receptor triggering, RAS binds GTP and forms heterodimers with the RAF protein kinases that directly phosphorylate and activate MEK1 and MEK2. Active MEK1/2 then phosphorylate and activate ERK1 and ERK2^9,10. Although multiple kinases can activate MEK1 and MEK2, they are the only kinases able to phosphorylate and activate ERK1/2. MEK1 and MEK2, act as gatekeepers and as such have been considered promising therapeutic targets in cancer and autoimmune diseases. The first MEK inhibitor was introduced more than 20 years ago. Since then, several new compounds have been developed and some of them are currently in phase III clinical trials as mono- or combined therapies in multiple cancers including melanoma, colorectal cancer and non-small-cell lung cancer^11,12. Although good results were obtained in solid cancers, they have given disappointing results in rheumatoid arthritis suggesting that MEK1/2 play more complex roles in the regulation of immune response¹³.

In mammals, MEK1 and MEK2 proteins are 80% identical¹⁴. The Mek1 null mutant dies at embryonic (E) day 10.5 of gestation from the underdevelopment of the extraembryonic tissues of the placenta^15,16,17. Conversely, Mek2^-/-mice are viable and fertile suggesting compensatory effects by Mek1¹⁸. The conditional deletion of Mek1 in the embryo proper generates viable and fertile Mek1^-/- mice indicating functional redundancy between Mek1 and Mek2 genes in embryo formation. This notion is further supported by the lack of obvious phenotypes in animals in which targeted tissues retain only one Mek1 or Mek2 functional allele^{19,20,21,22,23,24,25,26}. Indeed, developmental defects are only observed when all Mek1 and Mek2 alleles are deleted. More recently, we have shown that the MEK1 and MEK2 proteins are also functionally redundant for the development of the extraembryonic tissues. Thus, although MEK1 and MEK2 can substitute for each other, a minimum amount of MEK is critical for placenta development and embryo survival¹⁴.  

Several studies have established the crucial role of the ERK/MAPK pathway in hematopoietic cell proliferation and differentiation^4,5,6,7,8,27. ERK signaling is required for hematopoietic stem cell maintenance, in B and T cell positive selection as well as in B cell maturation^{5,28,29,30,31,32,33}. It is involved in B cell activation and survival following B cell receptor (BCR) and CD40 crosslinking^32,34,35. In addition, ERK pathway is active in the germinal center where activated B cells following T cell help proliferate, switch isotype class and increase the affinity of their BCR³⁰. However, some studies have described specific phenotype associated with the deletion of individual kinase of the ERK/MAPK pathway. The ablation of Erk1 increases splenic erythropoiesis and, impaired proliferation and maturation of thymocytes^7,36. The activation of ERK/MAPK pathway also plays a crucial role in regulating cytokine production by the antigen-presenting cells (APCs), namely the macrophages, dendritic cells (DC) and T cells^{37,38,39,40,41,42}. Perturbations of the ERK/MAPK pathway can modify the ratio of IL-10 and IL-12 and the differentiation of T helper (Th) cells^37,41,43,44. In Erk1^-/- mutants, changes in the IL-10/IL-12 ratio favor Th1 cell polarization over Th2 cell differentiation³⁷. Similarly, bone marrow derived macrophages (BMDM) isolated from Mek1 null mutants (Mek1^-/-;Tg^+/Sox2-cre) also showed a change in the IL-10/IL-12 ratio in response to TLR4 activation by the lipopolysaccharides (LPS), whereas MEK1/2 pathway inhibition or Mek1 deletion in BMDM enhance BMDM M2 polarization in response to IL-4/IL-13 stimulation^42,45. Together these data suggest that the ERK1/2 and MEK1/2 functional redundancy might be cell-type specific.

To specifically address the role of Mek1 and Mek2 in lymphoid and myeloid cell differentiation and function, and to circumvent the early embryonic lethality of Mek1 null mutants, the deletion of Mek1 function in a Mek2 null background was directed to the hematopoietic cell lineages using the Vav1-icre deleter mouse line. Mice without Mek1 and Mek2 genes function in the hematopoietic lineages were born with the expected Mendelian frequency, but they were anemic and died shortly after birth. Mice that retain one functional allele of Mek1 or Mek2 in the hematopoietic cell lineage, Mek1^+/flox Mek2^-/- Tg^+/Vav1-iCre (named hereafter 1Mek1) and Mek1^flox/flox Mek2^+/- Tg^+/Vav1-iCre (1Mek2) are viable and fertile. However, more than 50% of 1Mek1 and 1Mek2 females and 15% of the males died before 14 months of age. Both 1Mek1and 1Mek2 mutants present an almost normal erythropoiesis, but anomalies in the maturation and activation of their B and T cell lineages leading to autoantibody production against nuclear proteins and double stranded DNA, a hallmark of systemic lupus erythematosus (SLE). 1Mek1 mutants die from severe anemia and their necropsy showed a chronic glomerulonephritis associated with deposits of antibodies and complements leading most likely to kidney dysfunction severe life-threatening conditions reminiscent to condition seen in SLE patients. Thus, 1Mek1 mouse mutants provides a new genetic tool to address the role of the ERK/MAPK pathway in the immune response and in the pathogenic mechanisms of autoimmune disease.It also offers a unique model to characterize the molecular mechanisms underlying the pathogenesis of glomerulonephritis and to address the differences in SLE progression between females and males.

RESULTS

Loss of MEK function in hematopoietic cell lineages leads to death after birth due to severe anemia.

Ablation of Mek1 function in the hematopoietic cell lineages was produced with the Vav1-icre deleter mouse line on a Mek2 null background⁴⁶. Mek1^+/flox Mek2^-/- Tg^+/Vav1-iCre males were crossed with Mek1^flox/flox Mek2^-/- females to generate Mek1^flox/flox Mek2^-/- Tg^+/Vav1-icre embryos, thereafter, call Mek hematopoietic null (Mek^{hema null}) mutants. Expected Mendelian ratios were obtained at the different embryonic ages tested and at birth (Table S1). E16.5 Mek^{hema null}embryos and newborns appeared phenotypically normal compared to control littermates (Fig. 1A-C). Their vascularization and blood circulation did not present obvious defects at E16.5. However, even if Mek^{hema null}newborns did not show any sign of cyanosis at birth, they all died within the first 36 hours (Table S1). Their hematocrit was severely reduced as well as the size of their spleen suggesting defect in erythropoiesis and hematopoiesis (Fig. 1D-E).

To determine if the anemia and the reduction in size of the spleen in Mek^{hema null}mutants was due to defects in specific hematopoietic cell lineages, splenocytes from newborns were analyzed by flow cytometry using lineage-restricted markers. CD11b⁺ and Ly6G⁺ single positive and immature Ly 6G⁺CD11b⁺ double positive myeloid cell population as well as the CD19⁺ B cell population were drastically reduced in Mek^{hema null}mutants indicating a reduction in monocytes, macrophages, granulocytes, natural killer cell and B cell (Fig. 1F). If the data were normalized to correct for the reduced size of the spleen the reduction was even more severe.

Ter119 and CD71 staining and forward scatter (FSC) parameter was used to monitor the erythroblast subpopulations: proerythroblasts (ProE; Ter119^medCD71^highFSC^high), basophilic (Ery.A; Ter119^highCD7 ^highFSC ^high), late basophilic and polychromatic (Ery.B; Ter119^high CD71^highFSClow), and orthochromatic erythroblasts (Ery.C; Ter119^highCD7^lowFSC^low) in spleen and liver of litter matesfrom Mek1^+/flox Mek2^-/- Tg^+/Vav1-iCre x Mek1^flox/flox Mek2^-/- crossbreeding (Fig. 1G-H)^7,47. Ter119⁺ compartment had a trend to be reduced (data not shown) whereas the proportion of erythroblast in differentiation, namely the ProE and Ery.C, was significantly reduced in spleen of Mek^{hema null}mutants (3 to 4 times; Fig 1H). By contrast, the Ery.B cell population was increased. Altogether these results suggested a role for the ERK pathway in erythroid cell expansion and their differentiation, affecting the last steps of erythroid cell maturation.

Hematopoiesis occurs at sequential sites during embryogenesis migrating from the yolk sac (E7.5 to E 10.5), aorta, gonad and mesonephros (AGM) region (E8.5 to E11.5), the placenta E10.5 to E 13.5 and the liver (E10.5 to birth)⁴⁸. To determine whether the anemia and the decrease in total splenic cell number was due to a defect in the expansion of a specific progenitor, the total number of erythroid burst-forming unit (BFU-E, primitive erythroid progenitor), multipotent myeloid progenitor (CFU-GEMM) and granulocyte-macrophage progenitor (CFU-GM) colonies in the AGM at E10.5, the peripheral blood at E12.5 and the liver at E14.5 was monitored in methylcellulose colony-forming assays (Fig. 1I). No statistical differences were observed in the number of the different myeloid and erythroid progenitors generated by the control and Mek^{hema null} AGM. In contrast, no or few CFU-GM and CFU-GEMM colonies were obtained from peripheral blood and fetal liver from Mek^{hema null} embryos. BFU-E colony numbers were unaffected in the peripheral blood but reduced in the fetal liver. Altogether these results suggested that in absence of the ERK signaling, hematopoiesis was perturbed. At early time, before the action of the Cre recombinase, the progenitors were produced in the AGM allowing a first wave of erythropoiesis but at later stage when Mek1 and Mek2 genes were inactivated the expansion and/or the survival of the different myeloid cell progenitors is reduced and contributed to the anemia observed at birth⁴⁹.

Premature death of the 1Mek1 and 1Mek2 mutant mice

To assess the importance of the ERK pathway in hematopoiesis at later stages, Mek mutants were followed for survival rate over a period of 14 months. As shown in Fig. 2A and C, more than 60 and 80% of the female that retain one functional allele of Mek1 or Mek2 in the hematopoietic cell lineage, Mek1^+/flox Mek2^-/- Tg^+/Vav1-iCre (1Mek1) and Mek1^flox/flox Mek2^+/- Tg^+/Vav1-iCre (1Mek2) mutants died before 14 months of age compared to 20% and 50% for the male, whereas no dead animals were observed in the cohort of control littermate or wt. Some 1Mek1 and 1Mek2 mutants were found dead, but most of them were euthanized. For the 1Mek1 mutants, the euthanasia was mandatory because they were showing signs of anemia such as pale extremities, spiky hair and short of breath. Hematocrit was assessed at the time of euthanasia or on healthy 1Mek1 mutant animals at the end of the study. The hematocrit level was normal in the healthy 1Mek1 mutants but severely reduced in the mice euthanized (Fig. 2B). By contrast, in 1Mek2 mutants euthanized: five were weak and showed enlarge lymph nodes, six showed liver masses due to extramedullary hematopoiesis and one had dermatitis. Only 2 1Mek2 mutant presented signs of anemia as confirmed by its reduced hematocrit levels for the mutants analyzed (triangle in Fig. 2D). Moreover, 7 1Mek2 mutants were found death and their necropsy could not be performed. Even though both 1Mek1 and 1Mek2 mutants die prematurely, the causes of the death are distinct.

Defective hematopoiesis in 1Mek1 and 1Mek2 mutants

To determine if the 1Mek1 mutants were already presenting sign of anemia at earlier age, peripheral blood from 2- to 4-months old mutant mice was analyzed and compared to wt and single Mek null mutant: Mek1^flox/flox Tg^+/Sox2-cre (Mek1^-/-), Mek1^flox/flox Tg^+/Vav1-icre (Mek1^{hema null}) and Mek2^-/- mice. No difference in the hematologic parameters were observed between wt, Mek2^-/- and Mek1^{hema null} mutants (Fig. 2E-H). By contrast, Mek1^-/-,1Mek1 and 1Mek2 mutants showed a 10 to 13% reduction in red blood cell (RBC) number, hemoglobin concentration and hematocrit level indicating a trend to develop anemia. The number of circulating white blood cells in both 1Mek1 and 1Mek2 mutants was normal. Moreover, spleen weight/body weight ratio was twofold and threefold higher in 1Mek1 and 1Mek2 mutants, respectively (Fig. 2I-J). Proliferation index using phospho-histone H3 immunostaining revealed increased proliferation in 1Mek1 and 1Mek2 mutants red and white pulps indicating that the splenomegaly was due to defect in the expansion of erythroid and lymphoid cell lineages (Fig. 2K-L). Together, these results were suggesting that lymphopoiesis and erythropoiesis were affected in 1Mek1 and 1Mek2 mutants.

To determine whether the increase in 1Mek1 and 1Mek2 spleen size was due to the increase of a specific hematopoietic progenitor, colony forming cell assays in methylcellulose-based medium has performed. No statistical difference in progenitor numbers was observed in bone marrow of controls and 1Mek2 mutants. Only the number of multipotent CFU-GEMM and myeloid CFU-GM progenitors were reduced in 1Mek1 mutants (Fig. 3A). In spleen, the proportion of CFU-GEMM and BFU-E progenitors was reduced in 1Mek1; but their total number in the organ was not significantly reduced compared to controls (Fig. 3B). In contrast, the total number of CFU-GM, CFU-GEMM and BFU-E progenitors was increased in 1Mek2 mutants. Altogether, these results suggested that the hematopoietic progenitors were present in 1Mek1 and 1Mek2 mutants and their increased numbers in 1Mek2 mutant were actively contributing to the expansion of the spleen.

 

Impact of Mek mutation on erythropoiesis /

Normal response to erythropoietic stress signal in 1Mek1 and 1Mek2 mutants /

Normal erythropoiesis and response to erythropoietic stress in 1Mek1 and 1Mek2 mutants

Enlargement of the spleen in 1Mek1 and 1Mek2 mutants may be due to a hematologic stress response to produce more erythrocyte to compensate for the reduction of circulating RBCs^50,51. The increased proliferation observed in spleen red pulp supports this hypothesis. The moderate anemia and increased proliferation in the red pulp may also be induced to compensate to a reduction in erythrocyte survival. To assess the half-life of RBCs in the circulation, we measured the turnover of biotin-labeled RBCs by flow cytometry. 1Mek1 and 1Mek2 RBCs showed a half-life similar to wt and Mek1^-/- mutants (Fig. 3C). To directly test the ability of mutants to response to erythropoietic stress, phenylhydrazine (PHZ)-induced hemolytic anemia was used⁵⁰. Mice were injected with PHZ, and their hematocrit and reticulocyte counts were monitored over a period of 14 days (Fig. 3D). The hematocrits dropped sharply to 25% two days after PHZ treatment. Eight days later, all genotypes were returned to normal hematocrit levels. No significant difference was observed between control, 1Mek1 and 1Mek2 mutants (P = 0.96 by ANOVA). The fraction of peripheral blood reticulocytes was also used as indicator of erythropoietic response by FACS analysis (Fig. 3E). The basal reticulocyte count was below 2.5% in control,1Mek1 and 1Mek2 mutants. After PHZ-treatment, erythropoiesis is induced and reticulocyte count increased in all genotypes to reached maximal levels at day 6, no significant differences were detected between genotypes(P = 0.97 by ANOVA). The response of 1Mek1 and 1Mek2 mutants to erythropoietin was then tested. A single subcutaneous injection of EPO was made, and reticulocyte counts were monitored by FACS over a period of 10 days. Both controls and 1Mek1 and 1Mek2 mutants showed a similarly response (Fig. 3F; P = 0.97 by ANOVA).  Together, these results suggested that erythropoiesis in 1Mek1 and 1Mek2 mutants was not significantly affected and could hardly explain the development of the severe anemia leading to euthanasia in 1Mek1 mutants.

B and T cells maturation and activation defects in Mek mutants

In 1Mek1 and 1Mek2 mutants, Mek1 gene deletion with the Vav1-icre deleter mouse line also occurs in all lymphoid and myeloid lineages in a Mek2 null mutant background. Consequently, the Mek mutation can impact on leukocyte differentiation and homeostasis.  Both, 1Mek1 and 1Mek2 mutant spleens contained significantly increased numbers of myeloid cell population (Gr-1⁺, Mac-1⁺ and Gr-1⁺Mac-1⁺ sub population) as well as T (CD3⁺) and B (CD19⁺) cells indicating that the specific deletion of Mek1 and Mek2 or alternatively the  combine reduction of MEK protein were involved in these perturbations (Fig. 3G).

In order to determine at which step of myeloid or lymphoid cell differentiation the perturbation of the homeostasis takes place, a more systematic characterization of leukocytes differentiation and activation was first performed on Mek2^-/- and 1Mek1 littermates from Mek1^+/flox Mek2^-/- Tg^+/Vav1-iCre x Mek1^flox/flox Mek2^-/- breeding. Thymocytes from 1Mek1 mutants did not show statistical difference in the transition of the double negative T cell from DN1 to DN4 stage (CD25^–CD44⁺, CD25⁺CD44⁺, CD25⁺CD44^– and CD25^–CD44^–, respectively) when compare to Mek2^-/- control (Fig. S1A). Moreover, the  proportion of CD4⁺CD8⁺ double positive as well as  CD4⁺ and CD8⁺ single positive T cells were not statistically different (Fig. S1A). Similarly, the analysis of the B cells in the bone marrow (BM) did not reveal significant change in differentiation from pro-B (CD19⁺CD93⁺B220⁺c-kit⁺), pre-B (CD19⁺CD93⁺B220⁺c-kit^–IgM‑), immature recirculating B cell (CD19+CD93+B220+c-kit-IgM+) and plasmocytes (B220^–CD138⁺) between wt, Mek2^-/- and 1Mek1 mutants (Fig. S2). Moreover, no change in myeloid subpopulation: dendritic (CD11b^–Cd11c⁺), monocytes (CD11b⁺Cd11c^–) and mono-macrophages (CD11b⁺Cd11c⁺) was also observed in BM (Fig. S3).

In contrast, a major impact on the maturation and activation of B and T cells was observed in the 1Mek1 splenocytes. B and T cells compartment were also affected in 1Mek2 mutants splenocytes (Fig. 3G). Therefore,  we pursued our characterization of the splenocytes on an Mek1 Mek2 allelic series including Mek1^-/-, Mek2^-/-, 1Mek1, and 1Mek2 mutants and wt as control. The number of splenocytes were significantly increased in 1Mek2 mutants at 2.5 and 9 months, and in Mek1^-/- at 9-10 months and a trend to be increased in 1Mek1 at 9-10 months (P=0.072; Fig. 4A). Accordingly, the number of B cells (B220⁺CD19⁺CD93^–) was significantly increased in 1Mek2 mutants at 2.5 months and in Mek1^-/-, 1Mek1 and 1Mek2 mutants at 9-10 months (Fig. 4B). The immature transitional (B220⁺CD19⁺CD93⁺) and follicular (B220⁺CD19⁺CD93^–CD23⁺CD21^int) B cell numbers were also significantly increased in 1Mek2 mutants at 9-10 months and also at 2.5 months for the follicular B cells (Fig. 4C-D). The number of  germinal center B cell (B200⁺FAS⁺GL7⁺), enriched for autoreactive immature B cell (B220⁺CD19⁺CD93^–CD23^–CD21^–), plasma cells (B220^loCD138⁺) and follicular helper T (CD4⁺BCL6⁺PD⁺Foxp3^loCXCR5⁺) cells were increased in Mek1^-/-, 1Mek1 and 1Mek2 mutants at 2.5 and 9-10 months showing also a gradation according to the genotype (Fig. 4E-H). The increase number of germinal center B cells and plasma cells in 1Mek1 was indicating an augmentation of B cell activation which was confirmed by the expansion of the IgG1⁺ switched B cell population and the IgG1⁺ anticorps secreting cell numbers (Fig. 4I-J). In addition, the number of activated CD4+ and CD8+ T cells (CD44^hiCD62L^lo) were augmented in 2.5 months 1Mek1 mutant’s spleen and a trend to be increased or reduced was observed in bone marrow of Mek1^-/-, 1Mek1 and 1Mek2 mutants (Fig. 4K-L and Fig. S4). Finally as previously mention, the lymph nodes were often enlarged in euthanized 1Mek2 mutants and in specimens analyzed by FACS at 9-10 months of age (Fig. 5A).  However, the distribution of the B (B220⁺), CD4⁺ and CD8⁺ T cells were not change in 1Mek2 mutants (Fig5B-D). In contrast, the B cells population was increased in 1Mek1 mutants to the detriment of the CD4+ and CD8+ T cells. Together these results indicated that perturbation of the ERK pathway has major impact on B and T cell maturation and activation.

Defects in immune signaling pathway activation in Mek mutants

We have previously shown by the analysis of Mek1 Mek2 allelic series that the severity of placenta phenotypes correlated with the amount of MEK protein/activity independently of which MEK isoform was produced¹⁴. To determine if similar situation occur in hematopoiesis, we first assess the activation of the ERK pathway in response to immune stimuli in Mek1 Mek2 allelic series. B220⁺ splenocytes were stimulated with anti-IgG + IgM (H+L) and the kinetic of ERK activation was investigated by phospho-specific flow cytometry. We have previously shown that the Mek1 gene produces twice the amount of MEK protein than Mek2¹⁴. Consequently, ERK activation in B220⁺ splenocytes was proportional to the levels of the MEK proteins being maximal in wt and decreasing in Mek2^-/-, Mek1^-/-, 1Mek1 and be the lowest in 1Mek2 mutants (Fig. 6A). The reduction of the ERK pathway activation was associated to a decrease in the phosphorylation S6 ribosomal protein a downstream target of ERK (Fig. 6B). Surprisingly, the activation/phosphorylation of SYK and AKT were also reduced following the reduction of the ERK pathway activation (Fig. 6C-D).  Similarly, the activation of the ERK pathway in CD4⁺CD44⁺ Mek mutant T cells in response to anti-CD3 and -CD28 cross-linking treatment was also proportional to the levels of MEK proteins (Fig. 6E). A decrease in the phosphorylation of AKT was also observed in the Mek mutants (Fig. 6F). Together, these data indicated a gradient of ERK activation in this Mek1 Mek2 allelic series of Mek mutants in response to immune stimuli and affecting other signaling pathways which correlate with the immune phenotype observed and most likely contributing to the perturbation of the development of the immune system.

1Mek1 mutants develops a glomerulonephritis

The necropsy of the 1Mek1 mutants with severe anemia revealed the presence of pale kidney compared to reddish kidneys in control or healthy 1Mek1 mutants (Fig. 7). The histopathological analysis of the kidney using H&E and PAS staining showed evident renal injury characterized by mesangial proliferation with increased mesangial matrix, crescent formation and reduction of the Bowman space (Fig. 7C and F; black arrowhead, crescent). In addition, intracapillary with obliterated capillary lumens and thickened capillary walls was also observed. Tubular deterioration and dilation with hyaline casts were also noted (Fig. 7I; white arrowhead). In addition, the anemic 1Mek1 mutants showed markedly increased interstitial infiltration of immune cells suggesting inflammation (Fig. 7C and F). Presence of interstitial fibrosis was investigated by -SMA immunostaining. In control and healthy 1Mek1 mutants, anti--SMA stained the vascular smooth muscle lining the blood vessels. In anemic 1Mek1 mutant glomeruli with crescent, -SMA was detecting fibrocellular crescent indicating a glomerulonephritis (Fig. 7G-I; arrows, blood vessels; arrowheads, fibrocellular crescent). No significant glomerular lesions were seen in control and healthy 1Mek1 mutants (Fig. 7A-B and D-E). Immunofluorescent and immunohistochemistry staining revealed the presence of IgA, G, M and C3 complement deposition in the glomeruli of the 1Mek1 mutants (Fig. 7J-U and S5). Whereas no deposition of immunoglobulin or complement were detected in the glomeruli of the control or healthy1Mek1mutants. Together these results indicated a glomerulonephritis in the 1Mek1 mutants due to an autoimmune disorder.

In contrast, the necropsy of nine euthanized out of 15 1Mek2 mutants showed normal kidney histopathology at the exception of two specimens presenting moderate to severe anemia and glomerulonephritis (hematocrit lower to 40%; Fig. S4A-F). However, 4 out of the 15 1Mek2 mutants analyzed had infiltration of immune cells in the kidney and in the liver (3 euthanized and 1 healthy mutant; Fig. S4G, arrow).  Together, these data were suggesting that both 1Mek1 and 1Mek2 mutants have defects in the maturation and activation of its immune system but that only 1Mek1 are more susceptible to developed severe glomerulonephritis due to an autoimmune disease leading to inflammatory condition.

MEK reduced signaling leads to deregulated lymphopoiesis leading to autoimmunity

Autoimmune disorder such as systemic lupus erythematosus (SLE) generates glomerulonephritis due to the production of autoantibodies directed against nuclear and double stranded (ds) DNA with deposition of immunoglobulin and complement in the glomeruli and formation of hyaline cast. First, the presence of auto antibodies in the serum of 1Mek1 and 1Mek2 mutants was evaluated using the antinuclear antibodies (ANA) assay (Fig. 8A). No autoantibodies directed against nuclear protein were detected in the control serum whereas 15 out of 16 and 12 out of 13 serums from the euthanized and healthy1Mek1 mutants were positive for the ANA test, respectively. All serums tested for the1Mek2 mutants were also positive (8 serums from euthanized and 3 from healthy mutants). The presence of antibodies against double stranded DNA was also tested by ELISA. Significant higher anti-dsDNA titer were observed in both 1Mek1 and 1Mek2 mutants (Fig 8B).

B and T cells activation

Autoantibodies

MEK1 haplo-insufficiency leads to spontaneous autoantibody production

ELISA:

1. dsDNA:   of the anti-dsDNA antibodies in Mek1^-/-, 1Mek1 and 1Mek1 in 9 months mutants

ELISpot:

1.  of the number of dsDNA-specific IgG1⁺ secreting cells in Mek1^-/- and 1Mek1. Most likely also in 1Mek2, but the ELISpot did not work. Whereas

 

Phenotype increasing with the reduction in the activation of the ERK pathway

DISCUSSION

Our studies on the MEK isoforms has revealed unique functions as reflected by the distinct phenotypes of the Mek1 and Mek2 single mutants ^17,18,54. This specificity can be explained by the exclusive roles and/or the different spatial distribution or levels of MEK proteins ¹⁴. Our genetic studies on Mek gene function in hematopoiesis demonstrated that the presence of only one functional Mek allele in the hematopoietic cell lineages is sufficient to support normal development blood cellular compartment and to allow survival of mutant pups. Thus, Mek1 and Mek2 genes display redundant functions during the hematopoietic differentiation process. The phenotypes resulting from the ablation of Mek genes in the hematopoietic progenitors were expected due to the large spectrum of actions of the ligands mediating their action via the ERK/MAPK pathway.

 In this study, we described the generation of a hypermorphic

Our study provides genetic evidence for MEK1 and MEK2 functional redundancy in development. 

 In our studies, we demonstrated a novel role for the MEK1/2 pathway 

ff–v:

1. The Vav1-icre acts early during hematopoiesis before the generation of the definitive myeloid and lymphoid lineages ⁴⁶. However, the vav1 gene expression initiates around E11.5 in the liver. Thus, the expression of the Vav1 start in the liver when the liver begun to replace the yolk sac as the major hematopoietic organ and is composed very largely of the definitive (enucleated) erythrocytes.
2. The primitive erythropoiesis occurs normally before the deletion of Mek1 in the hematopoietic compartment via the Vav1-iCre transgene allowing survival during gestation, but lack of erythrocyte renewal leads to severe anemia around birth and neonatal death. 
3. any sign of cyanosis at birth, they all died in the first 36 hours => septicemie?

+f–v et ff+-v:

4. Premature dead due to anemia but no defects in erythropoiesis. The severe deterioration of kidneys in mutants can provoke a lack of erythropoietin production, which may explain the serious anemia observed.
5. This phenotype may impact on erythropoietin production by the kidney peritubular cells and cause the observed anemia.
6. The hematologic parameters were first monitored in mice carrying the Vav1-icre transgene as activity of the CRE recombinase can be in some condition deleterious causing excessive apoptosis ⁵⁵. The red blood cell, the hemoglobin concentration and the hematocrit level were reduced in Tg^+/Vav1-icre mutants, but leading only to a ~5% reductions (Table 3). In addition, the number of white blood cells were reduced by 40% and lymphocytes were reduced by half. By contrast, no statistical change were observed for the Mek1^flox-flox;Tg^+/Vav1-ice or in Mek2^-/- mutants.

METHODS

Mouse strains, genotyping and tissue collection  

The Mek1flox/flox and Mek2-/- mutant mouse lines and the   Sox2-cre and Vav-icre deleter mouse lines were previously described 17,18,46,56. The mouse line was maintained in the 129S6 background. The age of the embryos was estimated by considering the morning of the day of the vaginal plug as E0.5. Genomic DNA from embryonic yolk sacs and mouse tail biopsies was extracted, purified and genotyped by Southern blot and PCR analyses as previously described ^16,46. For RNA and protein extraction, organs were snap-frozen in N₂. Experiments were performed according to the guidelines of the Canadian Council on Animal Care and approved by the institutional animal care committee.

Hematologic parameters

Blood cell counts were analyzed with a UniCel DxC 600 Synchron Clinical Systems (Beckman Coulter). Hematocrit was assessed on a hematocrit centrifuge (IEC Micro MB centrifuge). The reticulocyte counts were measured as previously described ⁷. Briefly, 2 µl of whole blood was incubated with thiazole orange at 10^-4 mg/mL in phosphate-buffered saline (PBS) for 1 hour. Blood sample diluted in PBS was used as an unstained control. The reticulocyte counts were performed by FACS.

 

Hematopoietic cell preparations and Fluorescence-activated Cell Sorting Analysis

Single cell suspensions were prepared from mouse hematopoietic tissues by sieving and gentle pipetting through 70-μm nylon mesh. Peripheral blood cells and single-cell suspensions (10⁶) were saturated with Fc Block before staining. Stainings were performed with the following antibodies: anti-CD138 APC (281-2), anti-CD19 FITC (eBio1D3), anti-B220 Pacific Blue (RA3-6B2), anti-CD38 PE (90), anti-CD43 biotin (ebioR2-60), anti-CXCR4 PE (2B11), anti-CXCR3 biotin (CXCR3-173), and streptavidin Pe-Cy7 anti-Ter119, -CD4, -CD8, -CD19, -CD71, -Mac-1 (CD11b) and -Gr-1 (Ly6 G/C) antibodies by standard procedures ^18,57,58. FACS analysis was performed on a BD LSRFortessa cell analyzer.

 

Colony-forming assays

Methylcellulose colony-forming assays were performed in MethoCult M3434 complete medium with recombinant cytokines (StemCell Technologies). In brief, 2 x 10⁴ and 1 x 10⁵ cells from adultbone marrow and spleen, 2 x 10⁴ peripheral blood cells from E12.5 and fetal liver cells from E14.5 embryos and aorta-gonad mesonephros (AGM) region from E8.5 embryos were plated for the CFU assay. The cultures were incubated at 37°C in 5% CO2 for 2 days for CFU-E and 7 days for both colony-forming cell (CFC), BFU-E, and granulocyte-macrophage colony-forming unit (CFU-GM) colonies. All of the cultures were done in duplicate. Data as presented are mean ± SEM and are the result of three to eight independent specimens analyzed.

Histology, IHC and immunofluorescence (IF) analyses

Specimens were collected and processed for paraffin (5 μm) or frozen (6 μm) sections as described (Bissonauth et al., 2006). Kidney sections were stained with Hematoxylin and Eosin (H&E) and Periodic Acid-Schiff (PAS) staining according to standard histological procedures. IHC and IF experiments were performed as previously described ^15,16. Horseradish peroxidase activity was detected with the diaminobenzidine reagent kit (Zymed Laboratories). For IHC, slides were counterstained with hematoxylin.  For co-IF analyses, nuclei were visualized by DAPI staining.

Proliferation index was measured by counting the number of pHH3-immunoreactive cells, divided by the total cell number for each section analyzed from 2- to 4-months old mice. Eight random fields were analyzed for an average number of 500 cells per field. Four specimens per genotype were analyzed.

The primary antibodies used were: a rabbit monoclonal antibody against pHH3 (1/200; Cell Signaling), a rabbit polyclonal against SMA (1/200 dilution; Abcam), a Biotin-SP-conjugated goat anti-mouse IgG heavy and light chain (1/2000; Jackson ImmunoResearch Laboratories), a rat monoclonal antibodiy against complement C3 (1/50; Abcam) and a FITC-conjugated goat anti-mouse IgG/IgA/IgM heavy and light chain (1/250; Jackson ImmunoResearch Laboratories). The biotinylated secondary antibody was: a goat anti-rabbit (1/250; Vector Laboratories). The fluorescent secondary antibody was: a Cy5-conjugated goat anti-rat (1/250; Molecular Probes).

Erythrocyte half life

Erythrocyte half-life was evaluated using biotinylation of the entire blood cells and monitoring for cell replacement ⁵⁹. Biotinylation of blood cell was carried out by intravenous injection of 150 µl of 3.25 mg/ml of N-Hydroxysulfosuccinimide (NHS) esters (EZ-Link NHS-Biotin reagents Thermo Fisher Scientific, Waltham, MA) for two consecutive days. RBCs (3 x 10⁶) obtained from 1–5 µl of medial saphenous vein blood were labeled with 1µg de Strepavidin-AlexaFluor647-R-PE (Thermo Fisher Scientific, Waltham, MA) in 1 ml of PBS. The number of biotinylated cells in circulating blood was monitored at regular intervals by flow cytometry on a Coulter EPICS XL-MCL flow cytometer and Expo32 software (Beckman Coulter, Brea, CA)..

Phenylhydrazine stress test and response to erythropoietin induction

Mice were injected subcutaneously with 80 mg/kg phenylhydrazine hydrochloride (PHZ) solution in PBS at day 0 and 40 mg/Kg at day 1. Erythropoietin (EPO) induction was done by subcutaneous injection of 50U of Human recombinant EPO (Eprex) at day 0. Blood was obtained via the medial saphenous vein at regular intervals for hematocrit and/or reticulocyte count measurements. Data as presented are mean ± SEM and are the result of 4 to 5 independent specimens analyzed.

Anti-nuclear antibody (ANA) and Anti-dsDNA assays

HEp-2 anti-nuclear antibody (ANA) assays were performed as described ⁶⁰. Sera from mice were diluted (1:80) in PBS and incubated with HEp-2 cell substrate slides (Bio-Rad Laboratories, Montréal, QC) in a covered, humidified chamber for 30 min at RT°C. Slides were then rinsed twice in PBS for 5 min, and antibody binding was detected with the use of AlexaFluo 594-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific, Rockford, IL) at 1:250 for 30 min at RT°C. Nuclei were visualized by DAPI staining. Slides were next washed in PBS and mounted with coverslips for fluorescent microscopy with a Leica DM5500 B microscope. Mouse anti-dsDNA IgG antibodies were measured by ELISA according to the manufacturer’s instructions (Alpha Diagnostic ELISA kit; San Antonio, TX).

ANAs and ELISA

Antinuclear antibodies (ANAs) were detected by immunofluores- cence using Hep-2 slides (Cambridge Bioscience, UK). Hep-2 slides were first incubated for 30 minutes with serial dilutions of serum (from 1/50 to 1/200) and then washed and ANAs were detected with a rabbit anti-mouse Ig (H  L) secondary antibody conjugated with Alexa Fluor 488. Nuclei were counterstained with 4,6-diamidino-2- phenylindole.

Serum dsDNA-specific IgG was detected by ELISA. Briefly, ELISA plates were precoated with poly-L-lysine (Sigma-Aldrich) at 20 g/ml for 3 hours at room temperature before being coated with 20 g/ml of calf thymus dsDNA (Sigma-Aldrich) overnight at 4°C. Plates were saturated in PBS/2% BSA at 37°C for 2 hours. Sera were then added at a starting concentration of 1/100, serially diluted, and incubated for 2 hours at 37°C. Bound immunoglobulins were detected with a goat anti-mouse IgG Fc–specific conjugated with HRP (Jackson Immu- noresearch) and revealed with TMB (BD Biosciences). Pooled serum from six NZB/W mice was used as a standard.

ELISPOT

For detection of total IgG-secreting plasma cells, ELISPOT plates (Millipore) were coated with a mix of goat anti-mouse IgG1 + IgG2b  IgG2a + IgG3 antibodies (all at 2 g/ml) (Southern Biotech) over- night at 4°C.

For detection of dsDNA-specific antibody–secreting cells, ELISPOT plates were precoated with 20 g/ml poly-L-lysine (Sigma- Aldrich). The precoated plates were subsequently coated with calf thymus dsDNA (20 g/ml) (Sigma-Aldrich) and incubated over- night at 4°C.

Kidneys were first digested 30 minutes with collagenase (1 mg/ml) and DNase (100 ng/ml). Single-cell suspensions of kidneys and spleens were then obtained by homogenization on 70-m cell strainers (BD Bioscience) with cell culture medium (DMEM with 10% [vol/ vol] FCS, penicillin [100 units per ml], and streptomycin [100 g/ ml]; all from Life Technologies). Bone marrow was flushed from the bone and passed through a 70- m cell strainer. Cells were added to

saturated ELISPOT plates at 105 cells per ml and 106 cells per ml in quadruplicate and incubated overnight at 37°C in 5% CO2 in a humidified incubator with culture medium. Antibody-forming cells were detected with a goat anti-mouse IgG antibody adsorbed against other species and conjugated to horseradish peroxidase (Southern Biotech). Plates were developed using 3-amino-9-ethylcarbazole (Sig- ma-Aldrich). Plates were read using an AID ELISPOT reader accord- ing to the manufacturer’s instructions. Absolute numbers were mul- tiplied by 2 for the kidneys and by 7.9 for the bone marrow to extrapolate the total number of plasma cells in both kidneys and in total bone marrow.32

Statistical analyses

Samples were statistically compared using the Chi-squared test, Student’s t-test, the log-rank test and the Analysis of Variance on linear models (ANOVA), when appropriate. P < 0.05 was considered statistically significant.

 

FIGURE LEGENDS

Figure 1. Mice with no Mek function in their hematopoietic cell lineages die shortly after birth.

(A-B) Gross morphology of E16.5 and D0 control and Mek1^flox/flox Mek2^-/- Tg^+/Vav1-iCre embryos and newborn mice. Normal vascularisation and blood circulation was seen in controls and Mek1^flox/flox Mek2^-/- Tg^+/Vav1-iCre embryos at E16.5. (C) At birth no sign of cyanosis was detected at birth but the embryos die in the first 36 hours. (D) Macroscopic views showing the hypoplastic Mek1^flox/flox Mek2^-/- Tg^+/Vav1-iCre spleen phenotype at D1 compared with spleens from littermate controls. (E) The hematocrit was significantly decreased in D1 Mek1^flox/flox Mek2^-/- Tg^+/Vav1-iCre mice.

Figure 2. Flow cytometry analysis of splenic cells from Controls, Mek1^+/flox;Mek2^-/-;Tg^+/Vav1-icre and  Mek1^flox/flox;Mek2^-/-;Tg^+/Vav1-icre newborn mice.

Myeloid cell development was affected in Mek1^flox/flox Mek2^-/- Tg ^+/VaviCre mice, as shown by the reduced number of CD11b and Ly6 G/Csingle positive or double positive staining. B cell development was also affected in Mek1^flox/flox Mek2^-/- Tg ^+/VaviCre mice, the number of CD19 positive cells was decreased compared to control animals. Data as presented are mean ± SEM and are the result of five to 10 independent specimens analyzed (Controls, n=10; Mek1^+/flox Mek2^-/- Tg ^+/VaviCre, n= 8; Mek1^flox/flox Mek2^-/- Tg ^+/VaviCre, n=5).

Figure 3. Loss of erythroid and myeloid cell progenitors in Mek1^+/flox Mek2^-/- Tg^+/VaviCre embryos during gestation.

Number of CFU-GM, CFU-GEMM and BFU-E colonies obtained after in vitro growth of E10.5 aorta, gonad and mesonephros (AGM) region (A), and 2 x 10⁴ E12.5 peripheral blood cells (B) andE14.5 fetal liver cells (C) from controls and Mek1^+/flox Mek2^-/- Tg^+/VaviCre embryos. Data as presented are mean ± SEM and are the result of number of independent specimens indicated at the top of the graph. STAT Normal numbers of progenitors were observed at E10.5 before the action of the CRE recombinase. The Vav1 expression starts at E11.5. The number of all progenitor tested were significantly reduced at E14.5 after the CRE recombination.

Figure 4. Premature death of the Mek1^+/flox Mek2^-/- Tg ^+/VaviCre females.

(A, C) Kaplan-Meier curves showing the differential survival rate of Mek1^+/flox;Mek2^-/-;Tg ^+/Vav1-icre (A) and Mek1^flox/flox;Mek2^+/-;Tg ^+/Vav1-icre (C) mutant females and males monitored for 14 months. The P value was calculated by using the log-rank test; n: number of mice analyzed per genotype. (B. D) Hematocrit measurement on healthy and euthanized Mek1^+/flox Mek2^-/- Tg ^+/VaviCre mutant females and males at 14 months of age for healthy mice and between 3 to 14 months for the euthanized animals.

Figure 5. The Half-life of the erythrocyte and blood progenitor populations are not modified in Mek1^+/flox Mek2^-/- Tg^+/VaviCre mutants.

(A) Intravenous injection of biotin-reactive agent in tail vein of mice served to monitor RBC survival at regular intervals (days). The rate of disappearance of biotinylated RBCs was quantified to determine the half-life of the cells. Three to seven mice were assessed for each genotype. No statistical difference was observed. (B) CFCs-GEMMs, CFU-GMs and BFU-Es were measured after 7 days in complete methylcellulose with cytokines. Data are mean colony numbers with SEMs from 7 wt, 4Mek1^flox/flox Tg^+/Sox2Cre, 4Mek1^+/flox Mek2^-/- Tg^+/VaviCreand 5 Mek1^flox/flox Mek2^-/- Tg^+/VaviCre. *, P <  0.05; **, P < 0.01; ***, P < 0.001.

Figure 6. Normal erythropoiesis in Mek1^+/flox Mek2^-/- Tg^+/VaviCre and Mek1^flox/flox Mek2^-/- Tg^+/VaviCre response to erythropoietic stress and growth factor.

(A) Ten wild-type and 10 mutant mice were injected with PHZ on days 0, and 1. Hematocrits (upper panel) and reticulocyte counts (lower panel) were assessed every two days for two weeks. Data are mean ± SEM. Statistical difference for both mutant lines is indicated above the curve whereas statistical difference only for Mek1^+/flox Mek2^-/- Tg^+/VaviCre mutant is indicated under the curve (*P < 0.05). However, no statistical difference between genotype were observed using ANOVA. (B) Effect of a single IV injection of EPO on reticulocyte counts in Mek1^+/flox Mek2^-/- Tg^+/VaviCre and  Mek1^flox/flox Mek2^-/- Tg^+/VaviCremutant mice. ICR mice (/group) received a single SC subcutaneous injection of EPO (Eprex; 50U/mouse). Blood samples were collected after 1, 3, 4, 5, 7, 9 and 11 days, and reticulocytes were counted. The results are presented as the mean ± SEM of the values observed in 4 wild-type and 4Mek1^flox/flox Tg^+/Sox2Cre, 5Mek1^+/flox Mek2^-/- Tg^+/VaviCreand 4 Mek1^flox/flox Mek2^-/- Tg^+/VaviCre. No statistical difference between genotype were observed using ANOVA.

Figure 7. Glomerulonephritis in Mek1^+/flox Mek2^-/- Tg^+/VaviCre mice.

Hematoxylin/eosin staining (A-C) and SMA IHC (D-F) on kidney sections from wt (A, D), Mek1^+/flox Mek2^-/- Tg^+/VaviCre healthy (B,E) and euthanized (C,F) mice. The euthanized anemic mutants present enlarged glomeruli and cysts with protein deposition (C) and fibrosis as shown by SMA staining of glomeruli (arrowheads). Arrows, blood vessels.

In the periodic acid-Schiff (PAS)- stained section, the kidney in the vehicle- treated mice revealed moderate glomerular hypercellularity without crescent formation. There were no significant tubulointerstitial changes. In contrast, the tempol-treated ani- mals exhibited severe glomerular injury, with large fibrocellular crescents. There were also significant tubular atrophy and interstitial fibrosis, with prominent interstitial inflamma- tory infiltrate (original magnification  400).

Figure 8.

Figure 9. Detection of IgG deposition in kidney of Mek^hema mice.

IgG immunostaining (A-D) of kidney sections from wt (A-B) and Mek^hema (C-D) mice at 4 (A), 7 (C) and 14 (B,D) months (M). The mouse in C was euthanized due to severe anemia. IgG positive staining is detected in C and D (brown staining). Black arrows, glomeruli.

Figure 10. Mek1^+/flox Mek2^-/-Tg^+/VaviCre mice develop auto-antibodies and anti-dsDNA over time.

(A) Immunofluorescence staining of Hep2 cells with sera from wt (at 4 and 14 months) and healthy and euthanized Mek1^+/flox Mek2^-/-Tg^+/VaviCre(age in months is indicated). (B) Detection of anti-dsDNA antibody by ELISA. Black diamond, serum from old wt mice (n=9); black dots, serum from old Mek1^+/floxMek2^-/-Tg^+/Vav1Cre mice (n=12) and red dots, serum from euthanized Mek1^+/floxMek2^-/-Tg^+/Vav1Cre mice (n=7). *** p< 0.005.

ACKNOWLEDGEMENTS

 We thank Dr. Lucie Jeannotte for critical comments, Dr. D. Dimitris Kioussis for Vav1-icre mouse line. This work was supported by CIHR (MOP-97801 to J.C.).

AUTHOR CONTRIBUTIONS

L. B., S.-P. F.-B., M. E. and J.C. designed experiments and interpreted the results. L. B., S.-P. F.-B., É. P., N. H., R. A. and S. R. performed the experiments. L. B., S.-P. F.-B. and J.C. analyzed the data and J. C. wrote the paper.

Author contributions S.P.-Q. and R.B. designed experiments and interpreted the results. S.P.-Q. executed most of the experiments. S.P.-Q., M.F.-C., W.L., F.L., I.G.-G., A.H. and R.B. dissected mouse tissues and performed the FACS, immunostainings, microscopy and phenotypic analysis of the different mouse and cell lines. S.P.-Q. and S.F.R. developed methods and ImageJ scripts for assisted or automatic image quantification and analysis. S.F.R. generated DNA constructs and partially edited the paper. V.C.-G., S.F.R., I.G.-G., M.B. and R.B. generated and analyzed the ES and endothelial cell lines. R.B. and S.P.-Q. wrote the paper.

COMPETING INTERESTS

The authors declare that they have no competing interests.

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