Mouse Model of a Human STAT4 Point Mutation That Predisposes to Disseminated Coccidiomycosis

Abstract

STAT4 plays a critical role in the generation of both innate and adaptive immune responses. In the absence of STAT4, Th1 responses, critical for resistance to fungal disease, do not occur. Infection with the dimorphic fungus, Coccidioides, is a major cause of community-acquired pneumonia in the endemic regions of Arizona and California. In some people and often for unknown reasons, coccidioidal infection results in hematogenous dissemination and progressive disease rather than the typical self-limited pneumonia. Members of three generations in a family developed disseminated coccidioidomycosis, prompting genetic investigation. All affected family members had a single heterozygous base change in STAT4, c.1877A>G, causing substitution of glycine for glutamate at AA626 (STAT4^E626G/+ ). A knockin mouse, heterozygous for the substitution, developed more severe experimental coccidioidomycosis than did wild-type mice. Stat4^E626G/+ T cells were deficient in production of IFN-γ after anti-CD3/CD28 stimulation. Spleen cells from Stat4^E626G mice showed defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early postinfection, mutant Stat4^E626G/+ mice failed to produce IFN-γ and related cytokines in the lung and to accumulate activated adaptive immune cells in mediastinal lymph nodes. Therefore, defective early induction of IFN-γ and adaptive responses by STAT4 prevents normal control of coccidioidomycosis in both mice and humans.

Fig. 1.

Family Pedigree (A) Proband is indicated with a star. All filled symbols were sequenced and had the E626G mutation in STAT4. Striped symbol was not available for sequencing but did develop DCM. (B) Summary of shared coding variants found through sequencing. (C) Sequence of the guide RNA, used to generate the E626G mutation in exon 21 of the mouse STAT4 gene. PAM sequence is in green. Oligo sequence shows the introduced SNPs, one for the intended change E626G, and one to destroy the PAM sequence and prevent re cutting, and also to introduce a RsaI restriction enzyme site, used for genotyping. (D) Actual sequence chromatogram result showing the introduced by CRISPR/Cas9 homology-directed repair SNPs in a heterozygous founder.

Fig. 2.

Mice carrying Stat4 mutations show increased susceptibility to Coccidioides infection. (A) C57BL/6NJ (black lines), B6-Stat4^E626G/+ (red lines), or B6-Stat4^E626G/E626G (blue lines) mice (N=7/group) were infected intranasally with ~50 CFU of Cp1038. Mice were followed for disease survival. (B) C57BL/6NJ (black lines), B6-Stat4^−/− (red lines), B6-Stat4^E626G/− (blue lines) or B6-Stat4^+/− (green lines) mice (N=7/group) were infected intranasally with ~50 CFU of Cp1038. Mice were followed for disease survival. Graph is representative of 3 separate experiments of similar design. Significance was determined using a Mantel-Cox Log-rank test.

Fig. 3.

CD4+ T cells were purified from C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares), or B6-Stat4^−/− (blue triangles) spleens. Purified cells were then plated with rmIL-2 and activated with CD3/28 Dynabeads according to the manufacturer’s instructions for 48 hours. At this point cells were supplemented with the indicated cytokine for 24 more hours. PMA/Ionomycin samples were treated for the last 4 hours of the experiment. Supernatants were then collected and analyzed for IFN-gamma production by ELISA. Graph is representative of 2 separate experiments of similar design. Significance was determined using a Mann-Whitney.

Fig. 4.

Stat4^E626G/+ mice have similar cellularity in the lungs and mediastinal lymph nodes after Cp1038 infection. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time points mice were sacrificed (N=4–5/time point) and total cellularity was determined in the lungs (A) mediastinal lymph nodes (B) and spleen (C). Graph is representative of 2 separate experiments of similar design. Significance was determined using a Mann-Whitney on log transformed data.

Fig. 5.

Stat4^E626G/+ mice have slightly decreased accumulation of B cells the lungs after Cp1038 infection. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038. At 2 weeks post infection mice were sacrificed (N=4–5/group) and total numbers of T (CD3⁺ CD19⁻)(A) and B (CD19⁺ B220⁺)(B) cells were determined in the lungs by flow cytometry. B cells were further subtyped by expression of IgM and IgD (C-F). Significance was determined by Mann-Whitney on log transformed data. Data is combined from 2 experiments of similar design.

Fig. 6.

Stat4^E626G/+ mice have decreased accumulation of total and activated T in the mediastinal lymph nodes 2 weeks after Cp1038 infection. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038. Two weeks after infection mice were sacrificed (N=4–5/time point) and T cells were phenotyped in the mediastinal lymph nodes. (A) Total T Cells (CD3+ CD19−). (B,E) Naïve T cells (CD62L+ CD44var), (C,F) Effector T Cells (CD62L− CD44−), (D,G) Memory T Cells (CD62L− CD44+). Significance was determined by Mann-Whitney of log transformed data. Data is combined from 2 experiments of similar design.

Fig. 7.

Stat4^E626G/+ mice have decreased accumulation of total and activated B cells in the mediastinal lymph nodes 2 weeks after Cp1038 infection. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038. Two weeks after infection mice were sacrificed (N=4–5/time point) and total B cells (CD19+ B220+) were enumerated in the mediastinal lymph nodes (A). B cell were further phenotyped for their expression of IgM and IgD (B-E). Significance was determined by Mann-Whitney of log transformed data. Data is combined from 2 experiments of similar design.

Fig. 8.

Stat4^E626G/+ mice have decreased accumulation of total and activated CD4+ T cells in the mediastinal lymph nodes early after Cp1038 infection. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time point after infection mice were sacrificed (N=4–5/time point) and total CD4+ T cells were enumerated in the mediastinal lymph nodes (A). Cells were further divided into naïve (B) (CD62L+, CD44var), Effector (C) (CD62L− CD44−), and Memory (D) (CD62L− CD44+) subtypes. Significance was determined by Mann-Whitney of log transformed data. Data is combined from 2 experiments of similar design.

Fig. 9.

Stat4^E626G/+ mice have decreased accumulation of total and activated CD8+ T cells in the mediastinal lymph nodes early after Cp1038 infection. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time point after infection mice were sacrificed (N=4–5/time point) and total CD8+ cells were enumerated in the mediastinal lymph nodes (A). Cells were further divided into naïve (B) (CD62L+, CD44var), Effector (C) (CD62L− CD44−), and Memory (D) (CD62L− CD44+) subtypes. Significance was determined by Mann-Whitney of log transformed data. Data is combined from 2 experiments of similar design.

Fig. 10.

Stat4^E626G/+ mice have decreased accumulation of NK and ILC1 cells in the mediastinal lymph nodes early after Cp1038 infection. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time point after infection mice were sacrificed (N=3–5/time point) and total NK cells (NK1.1+, NKp46+, CD49a−) (A) and Type 1 ILCs (NK1.1+, NKp46+, CD49a+) were enumerated in the mediastinal lymph nodes. Significance was determined by Mann-Whitney of log transformed data.

Fig. 11.

Stat4^E626G/+ mice have similar fungal burdens after Cp1038 infection as compared to B6 mice. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038 at the indicated time point after infection mice were sacrificed (N=4–5/time point) and fungal burdens were determined in the lungs (A) and spleen (B). Significance was determined by Mann-Whitney of log transformed data. Data in representative of 2 experiments of similar design.

Fig. 12.

Stat4^E626G/+ mice have decreased production of IFN-gamma and related cytokines after Cp1038 infection as compared to B6 mice. C57BL/6NJ (black circles), B6-Stat4^E626G/+ (red squares) mice were intranasally infected with Cp1038 at the indicated time point after infection mice were sacrificed (N=4–5/time point). Lungs were finely sliced and placed in complete RPMI for 24 hours. Supernatant was then collected and analyzed for IFN-gamma (A), IP-10 (B), and MIG (C) cytokine production by ELISA. Serum was harvested and analyzed for IFN-gamma (D), IP-10 (E), and MIG (F) by ELISA. Significance was determined by Mann-Whitney.