Murine models of Pneumocystis infection recapitulate human primary immune disorders

Abstract

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Keywords: Cytokines; Fungal infections; Immunology; Infectious disease; T cells.

Figure 1. CD4⁺ T cell STAT3 signaling is required for Pneumocystis clearance.

For primary infection model, WT and KO mice were infected for 4 weeks with 2 × 10⁵ P. murina asci. (A) Schematic timeline of primary infection model. (B–E) Real-time PCR of whole lung RNA for P. murina mitochondrial ribosomal RNA large subunit (LSU) was performed and quantified to assess degree of P. murina burden. SCID, severe combined immunodeficiency; DKO, double KO; TKO, triple KO. For the T cell intrinsic model, WT and KO CD4⁺ T cells were adoptively transferred via i.v. injection to Rag1^–/– mice 2 weeks prior to primary infection. (F) Schematic timeline of T cell intrinsic model. (G–I) Real-time PCR of whole lung RNA for P. murina mitochondrial ribosomal RNA large subunit was performed and quantified to assess degree of P. murina burden, reported as means ± SEM for n = 4–6 per group. B and C were not repeated. D, E, and G–I are representatives of 2 experiments. P values are annotated as follows: **P ≤0.01, ***P ≤0.001, and ****P ≤0.0001 (1-way ANOVA).

Figure 2. CD4⁺ T cell IL-21 signaling is required for P. murina clearance.

WT and KO mice were infected for 4 weeks with 2 × 10⁵ P. murina asci. (A) Real-time PCR of whole lung RNA for P. murina mitochondrial ribosomal RNA large subunit was performed and quantified to assess degree of Pneumocystis burden. (B) Chitin-specific IgM antibodies in the serum were measured by direct ELISA against chitin at absorbance OD450. (C) Pneumocystis-specific IgG antibodies were measured by direct ELISA against P. murina at absorbance OD450. WT and KO CD4⁺ T cells were adoptively transferred via i.v. injection to Rag1^–/– mice 2 weeks prior to primary infection. (D) Real-time PCR of whole lung RNA for P. murina mitochondrial ribosomal RNA large subunit was performed and quantified to assess degree of Pneumocystis burden. Lungs were also digested into a cell suspension and stained with (E) anti-CD3 and anti-CD4 to define CD4⁺ T cells and (F) annexin V and PI for apoptotic cells. Values are reported as means ± SEM for n = 4–6 per group. A–F are representative data of 2 experiments. P values are annotated as follows: ***P ≤0.001, and ****P ≤0.0001 (1-way ANOVA).

Figure 3. IL-22–FC treatment is protective in Il21r^–/– mice.

(A) Histology slides of lung tissue from WT naive and P. murina–infected mice were stained for IL-22RA by IHC. IL-21R–deficient and CD4-depleted WT mice were infected with P. murina for 2 weeks prior to 2 weeks of biweekly treatment of IL-22–FC (n = 4). (B) Schematic timeline of IL-22–FC treatment model. Real-time PCR of whole lung RNA for (C) P. murina mitochondrial ribosomal RNA large subunit, (D) Sp, and (E) Arp was performed to assess degree of Pneumocystis burden. (F) Schematic timeline of the short-term IL-22–FC treatment model where IL-21R–deficient mice received a single dose of IL-22–FC at the 2 week mark of infection (n = 3). (G) Representative transmission electron microscopy images of pneumocyte/trophozoite interaction. (H) Quantification of percent trophozoite perimeter bound to murine pneumocytes. (I) Whole lung RNA was isolated, sequenced using an Illumina NextSeq 500, and analyzed for differential expression of genes associated with antimicrobial responses (n = 3). P. murina was cultured in vitro with 10 or 50 μg/ml concentrations of LL-37 protein. RNA from in vitro incubation with LL-37 was used to perform Real-time PCR on (J) P. murina mitochondrial ribosomal RNA small subunit, (K) Arp, and Sp and normalized to input expression. Values are represented as means ± SEM. A–E, J, and K are representative of 2 experiments. G–I were performed once. P values are annotated as follows: *P ≤0.05, **P ≤0.01, and ****P ≤0.0001 (1-way ANOVA).

Figure 4. GM-CSF production by CD4⁺ T cells is required for successful immune response.

WT and Csf2^–/– CD4⁺ T cells were adoptively transferred via i.v. injection to Rag1^–/– mice 2 weeks prior to primary infection. Real-time PCR of whole lung RNA for (A) P. murina mitochondrial ribosomal RNA small subunit was performed to assess degree of P. murina burden, as well as on murine expression markers Arg1 (B), Nos2 (C), Signr4 (D), and Mrc1 (E). (F) Whole lung RNA was isolated from WT and WT CD4-depleted mice, which were infected with P. murina for 2 weeks. RNA was then sequenced using an Illumina NextSeq 500 and analyzed for differential expression of Signr family members (n = 4). (G) Representative images of P. murina inoculum incubated directly with human CD209-FC (hIgG4) recombinant protein and stained with DAPI (blue) and anti-hIgG4 (FITC). Values are represented as means ± SEM. A is a composite of 2 experiments. B–E and G are representative data of 2 experiments. F was performed once. P values are annotated as follows: *P ≤0.05, **P ≤0.01, ***P ≤0.001, and ****P ≤0.0001 (1-way ANOVA).

Figure 5. Combinatorial IL-22 and GM-CSF treatment is protective in CD4-depleted mice.

CD4-depleted WT mice were infected with P. murina for 2 weeks prior to 2 weeks of biweekly treatment of IL-22–FC and triweekly administration of recombinant GM-CSF (n = 4). (A) Schematic timeline of IL-22–FC and GM-CSF combinatorial treatment model. Real-time PCR of whole lung RNA for (B) P. murina mitochondrial ribosomal RNA small subunit, (C) Arp, and (D) Sp was performed to assess degree of P. murina burden. P values are annotated as follows: *P ≤0.05 and **P ≤0.01 (1-way ANOVA).