1051 - Increased susceptibility to Streptococcus pneumoniae pulmonary infection in the Eµ-TCL1 adoptive transfer mouse model of CLL

Infectious complications continue to be a major cause of morbidity and mortality in patients with CLL. The reason of this increased risk is multifactorial and is associated with immunosuppression generated by treatment, but also by the disease itself. In fact, treatment-naïve patients are at higher risk of infections than healthy population, and infections during the treatment-naïve period are associated with worse treatment-free survival and overall survival (Andersen, M. A. et al. Haematologica 2018). Bacterial pneumonia, involving Streptococcus pneumoniae (Spn), is a frequent serious infection among untreated patients (Clare Sun, et al. Hematol Oncol Clin N Am. 2021). Early immune response to Spn is critical to control bacterial burden and disease outcome. Although innate immune alterations were described in CLL, if these mechanisms are defective in the Spn in vivo response is unknown. By using a murine adoptive transfer model of CLL, we aimed to study the susceptibility to Spn pulmonary infection in the context of untreated CLL and to characterize innate immune parameters involved in the early response to the bacteria.
Female C57BL/6 mice (8-10 weeks old) were intraperitoneally injected with 20 x 106 leukemic cells obtained from spleens of full leukemic Eµ-TCL1 mice (C57BL/6 background). CLL burden was determined in the adoptively transferred (AT)-TCL1 mice with the percentage of leukemic cells (CD5+ CD19+) in the peripheral blood (PB) assessed by flow cytometry (FC). Leukemic mice (> 60% of CD5+ CD19+ cells in PB) and control mice (age-matched C57BL/6) were intranasally (i.n.) infected with a total of 2 x 106 CFU of Spn serotype 3 (clinical isolate provided by the Antimicrobial Service from “Dr. Carlos G. Malbrán” Institute, Argentina) (Figure 1A). For survival experiments, mice were daily controlled for 11 days. In other set of experiments, bronchoalveolar lavage fluid (BALF) analysis from infected mice was performed. To this aim mice were euthanized 24 hs after infection and the BALF was obtained to determine: bacterial load by serial dilution in Columbia blood agar plates, cell number and phenotype by FC, IL-1β and TNF-α by ELISA and total protein, Lactate Dehydrogenase (LDH) and myeloperoxidase (MPO) by colorimetric assays. Statistical analysis was performed with Prism v7 (GraphPad). Values of p < 0.05 was considered significant.
Spn-infected AT-TCL1 mice showed an increased mortality rate compared to Spn-infected control mice and non-infected AT-TCL1 mice (Figure 1B). At 24hs after infection, AT-TCL1 mice showed a higher bacterial burden than control mice in the BALF, suggesting impairment in bacterial growth control-mechanisms (Figure 1C). We also observed higher levels of inflammatory parameters in BALF from leukemic mice such as total protein, TNF-α and IL-1β levels (Figure 1D-F). Interestingly we found no difference in LDH values between control and leukemic mice (Figure 1G), suggesting similar levels of lung damage in both cases. Moreover, we found similar numbers of neutrophils in the BALF of AT-TCL1 and control mice after 24hs of infection (Figure 1H). This is different from what was previously reported in a urinary tract infection model in AT-TCL1 where a defect in neutrophil recruitment to the infection site was found (Subramaniam, N et al. Blood Adv. 2021). Remarkably, despite a similar neutrophil-recruitment to the lung, AT-TCL1 mice showed lower levels of MPO in the BALF than control mice (Figure 1I).
Our results show that CLL predisposes to an impaired control of bacterial growth and to a higher mortality rate in a model of Spn pulmonary infection. The increased bacterial count in the BALF of AT-TCL1 mice was accompanied with a rise in inflammatory parameters, suggesting that at least some early innate-immune mechanisms, such as pro-inflammatory cytokines release and neutrophil recruitment, are preserved. On the other hand, and given the central role of MPO in bacterial clearance by neutrophils, the lower levels of MPO found in the BALF of AT-TCL1 infected mice, suggests that the increased susceptibility to Spn lung infection could be partially related to a deficiency in killing mechanism by neutrophils.