The Impact of Genetic Susceptibility to Systemic Lupus Erythematosus on Placental Malaria in Mice

<div><p>Severe malaria, including cerebral malaria (CM) and placental malaria (PM), have been recognized to have many of the features of uncontrolled inflammation. We recently showed that in mice genetic susceptibility to the lethal inflammatory autoimmune disease, systemic lupus erythematosus (SLE), conferred resistance to CM. Protection appeared to be mediated by immune mechanisms that allowed SLE-prone mice, prior to the onset of overt SLE symptoms, to better control their inflammatory response to <i>Plasmodium</i> infection. Here we extend these findings to ask does SLE susceptibility have 1) a cost to reproductive fitness and/or 2) an effect on PM in mice? The rates of conception for WT and SLE susceptible (SLE^s) mice were similar as were the number and viability of fetuses in pregnant WT and SLE^s mice indicating that SLE susceptibility does not have a reproductive cost. We found that <i>Plasmodium chabaudi</i> AS (<i>Pc)</i> infection disrupted early stages of pregnancy before the placenta was completely formed resulting in massive decidual necrosis 8 days after conception. <i>Pc</i>-infected pregnant SLE^s mice had significantly more fetuses (∼1.8 fold) but SLE did not significantly affect fetal viability in infected animals. This was despite the fact that <i>Pc</i>-infected pregnant SLE^s mice had more severe symptoms of malaria as compared to <i>Pc</i>-infected pregnant WT mice. Thus, although SLE susceptibility was not protective in PM in mice it also did not have a negative impact on reproductive fitness.</p></div

High dose dasatinib treatment markedly reduces the numbers of B-lineage cells.

<p>c-Cbl RING finger mutant mice aged 8–9 months were dosed daily with 30 mg/kg (am) +50 mg/kg (pm) of dasatinib or vehicle, and analyzed after 4 weeks. (A) Numbers of nucleated bone marrow cells from each group. (B) Bone marrow cells analyzed by flow cytometry to determine the percentage of CD11b⁺ myeloid-lineage cells; CD19⁺ B-lineage cells; lineage negative cells (Lin⁻); c-Kit⁺ lineage negative (LK) cells; Lin⁻ Sca-1⁺ c-Kit⁺ (LSK) cells; and multi-potent progenitors (MPPs). (C) Spleen weights from each treatment group. (D) Spleen cells analyzed by flow cytometry to determine percentages of CD11b⁺, CD19⁺ and T cell receptor⁺ (TCR) cells. All of the above data are from 4 dasatinib and 4 vehicle treated mice. The results are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 using the unpaired Student’s <i>t</i> test. (E) H&E stained sections of spleens showing the effects of dasatinib in markedly reducing the size of the follicles in the white pulp. The images were acquired at room temperature using an Olympus BX51 microscope. Photomicrographs were taken at 40× and 200× magnification with 4×/0.16 and 20×/0.70 objective lenses using an Olympus DP70 digital camera. Scale bars are 2 mm and 200 μm respectively.</p

Dasatinib Targets B-Lineage Cells but Does Not Provide an Effective Therapy for Myeloproliferative Disease in c-Cbl RING Finger Mutant Mice

<div><p>This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the <i>in vitro</i> growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of <i>in vivo</i> effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.</p></div

Dasatinib targets immature and germinal center B cells.

<p>B6.CD45.1 mice repopulated with bone marrow cells from a c-Cbl^A/− mouse were dosed twice daily with 15 mg/kg of dasatinib for 16 days. WBCs and spleen cells were analyzed by flow cytometry to identify B cell populations that are most sensitive to dasatinib. Shown are representative flow cytometry profiles and data from 3 vehicle and 3 dasatinib treated mice. (A) B220⁺ WBCs and (B) B220⁺ spleen cells were analyzed for the expression of IgM and IgD to identify immature and mature B cells. The gates show immature IgM⁺ IgD⁻ B cells. (C) B220⁺ spleen cells analyzed for the expression of GL7 and CD95 to identify germinal center (GC) B cells. The results are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01 using the unpaired Student’s <i>t</i> test.</p

High dose dasatinib treatment markedly reduces the numbers of B-lineage cells.

<p>c-Cbl RING finger mutant mice aged 8–9 months were dosed daily with 30 mg/kg (am) +50 mg/kg (pm) of dasatinib or vehicle, and analyzed after 4 weeks. (A) Numbers of nucleated bone marrow cells from each group. (B) Bone marrow cells analyzed by flow cytometry to determine the percentage of CD11b⁺ myeloid-lineage cells; CD19⁺ B-lineage cells; lineage negative cells (Lin⁻); c-Kit⁺ lineage negative (LK) cells; Lin⁻ Sca-1⁺ c-Kit⁺ (LSK) cells; and multi-potent progenitors (MPPs). (C) Spleen weights from each treatment group. (D) Spleen cells analyzed by flow cytometry to determine percentages of CD11b⁺, CD19⁺ and T cell receptor⁺ (TCR) cells. All of the above data are from 4 dasatinib and 4 vehicle treated mice. The results are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 using the unpaired Student’s <i>t</i> test. (E) H&E stained sections of spleens showing the effects of dasatinib in markedly reducing the size of the follicles in the white pulp. The images were acquired at room temperature using an Olympus BX51 microscope. Photomicrographs were taken at 40× and 200× magnification with 4×/0.16 and 20×/0.70 objective lenses using an Olympus DP70 digital camera. Scale bars are 2 mm and 200 μm respectively.</p

High dose dasatinib treatment results in a significant reduction of B lymphocytes in the blood.

<p>c-Cbl RING finger mutant mice aged 8–9 months were dosed daily with 30 mg/kg (am) +50 mg/kg (pm) of dasatinib or vehicle, and bled before treatment (Pre-Tx), and after 2 and 4 weeks of treatment. Differential blood counts from 9 vehicle and 7 dasatinib treated mice were determined by Hemavet analysis. Shown are (A) total WBC numbers and (B) lymphocyte numbers. (C) WBCs were analyzed by flow cytometry to determine the percentage of B-lineage cells, by anti-CD19 staining, and the percentage of T cells, by anti-T cell receptor staining. (D) Numbers of neutrophils and (E) monocytes. (F) Blood films from 2 vehicle and 2 dasatinib treated mice following 4 weeks of dosing illustrate the loss of lymphocytes in the dasatinib-treated mice. Lymphocytes are indicated by red arrows, and myeloid cells by black arrows, in the blood films from the two vehicle treated mice. The images were acquired at room temperature using an Olympus BX51 microscope with a 60×/0.09 objective and photographed with a SIS 3VCU Olympus digital camera. Scale bar = 50 μm. Lymphocyte numbers (G) and percentages (H) in the blood return to pre-treatment levels 4 weeks after ceasing dasatinib treatment. Neutrophil numbers remain unaltered (I) but the high percentages (J) returned to normal 4 weeks after dasatinib dosing ceases. Mice were bled from the tail vein before treatment (Pre-Tx), after 2 and 4 weeks of treatment, and following 4 weeks without treatment (i.e. post-Tx). Numbers and percentages were determined by Hemavet differential counting, and are from 4 vehicle and 3 dasatinib treated mice. The results are expressed as means ± standard errors. **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 using unpaired Student’s <i>t</i> test.</p

Dosing c-Cbl RING finger mutant mice with dasatinib results in a reduction of lymphocytes and a corresponding increase in neutrophils.

<p>c-Cbl^A/− mice aged 8–9 months were bled 6 days before dosing (Pre-Tx), and after 2 and 4 weeks of daily dosing with 15 mg/kg of dasatinib or vehicle. (A) Numbers of white blood cells (WBC), lymphocytes, neutrophils and monocytes. The counts are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, using unpaired Student’s <i>t</i> test. (B) WBC lysates prepared from mice dosed for 4 weeks with vehicle or dasatinib were immunoblotted with the indicated antibodies. (C) Analysis of mice after 4 weeks of dosing showing the numbers of nucleated bone marrow cells, (D) the proportion of CD11b⁺; CD19⁺; lineage negative (Lin⁻); Lin⁻, c-Kit⁺ (LK); Lin⁻, Sca-1⁺, c-Kit⁺ (LSK) cells; and multi-potent progenitors (MPPs: defined as FLT3⁺ LSK cells), expressed as percentages of the indicated populations. (E) Megakaryocyte erythroid progenitors (MEPs) and common lymphoid progenitors (CLPs), and (F) common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) expressed as percentages of Lin⁻ bone marrow cells. The bone marrow data is from 4 dasatinib and 4 vehicle treated mice, and the results are expressed as means ± standard errors. ***<i>P</i><0.001 using the unpaired Student’s <i>t</i> test. (G) Spleen weights from 4 vehicle and 4 dasatinib treated mice after 4 weeks of dosing.</p

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

<div><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.</p></div

Histopathology in the lungs following primary infection with EMC/2012 strain of MERS-CoV.

<p>Images show H&E (left) and IHC against the MERS-CoV N protein (right) following infection with 10⁵ TCID₅₀ (A,D), 10³ TCID₅₀ (B,E), or a media only control (C,F). All images at 10x, (bar equivalent to 100μm) with 40x insets (bar equivalent to 20μm). Images shown are from day 3 post-infection for all groups.</p

Detection of complement protein during primary infection and reinfection.

<p>ELISA against C3a protein in rabbit lung homogenates (A) show an increase in C3a levels present during reinfection compared to primary infection. Immunofluorescence images show MERS-CoV N antigen (green) and complement (red) following secondary infection (B) and primary infection (C). Images from day 3 post-infection at 40x, bar equivalent to 20μm. n = 3 rabbits per group. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. p values *<0.05.</p