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 (STAT4E626G/+). A knockin mouse, heterozygous for the substitution, developed more severe experimental coccidioidomycosis than did wild-type mice. Stat4E626G/+ T cells were deficient in production of IFN-γ after anti-CD3/CD28 stimulation. Spleen cells from Stat4E626G mice showed defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early postinfection, mutant Stat4E626G/+ 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.

STAT4 is critical in the generation of the Th1 response, which is mediated in part by IL-12 and IL-18. Mice deficient in STAT4 do not make effective Th1 responses and are more susceptible to infections requiring effective IFN-γ responses (17). In mice many polymorphisms of STAT4 are known but there are few data available on the impact of those substitutions (8). In humans, the IL-12 axis has been implicated in autoimmunity as well as infections, including paracoccidioidomycosis (4, 915).

Coccidioidomycosis (CM), also known as San Joaquin Valley fever, is caused by Coccidioides immitis and Coccidioides posadasii, soil-inhabiting dimorphic fungi in endemic regions of the Southwestern United States, Mexico, and other parts of the Western Hemisphere. Despite its relatively limited geographic distribution, at least 150,000 infections occur annually (16). Recent estimates from the Centers for Disease Control and Prevention propose that the actual numbers could be several-fold higher (17). Economic estimates for the health-related cost of CM in California and Arizona total $1.4 billion/year (18, 19).

Most infections are either asymptomatic or self-limited, although the latter often cause significant illness for months. Within endemic areas of Arizona, CM accounts for a quarter of all community-acquired pneumonias (20, 21). In ∼0.5–1.0%, of all CM infections, extrapulmonary dissemination leads to progressive and destructive lesions in other organs, a complication known as disseminated CM (DCM). DCM is much more likely in patients with broadly depressed cellular immunity, either from comorbidities or immunosuppressive therapies (22). However, most DCM patients do not have these risk factors (23, 24), leaving the immunologic basis for their progressive infections unclear.

We have previously investigated patients with DCM and found mutations in the IL-12/IFN-γ pathway. These have been isolated patients or siblings with DCM, harboring deleterious mutations in IL12RB1, IFNGR1, STAT1, and STAT3 (2529). Consistent with the above findings, IFN-γ knockout mice have higher fungal burdens, especially in disseminated sites, than do wild-type (WT) mice (30). Similarly, it is clear that transfer of immunity following vaccination requires IFN-γ–competent CD4+ T cells (30). Therefore, the IL-12/IL-18/STAT4/IFN-γ axis appears to be essential for protection from DCM in both humans and experimental animals.

We have identified members of a three-generation family who developed DCM, all of whom possessed a shared, unique single amino acid missense mutation in STAT4. We investigated the consequences of that mutation in vitro, genetically engineered mice carrying the homologous mutation, experimentally infected those mice, and examined the potency of a CM vaccine in them. The induced mutation impaired IFN-γ production through STAT4-dependent agonists, leading to a failure to accumulate activated immune cells in the draining lymph nodes, shorter survival time, and increased Coccidioides fungal burden.

Peripheral blood was used for sequencing after written consent under National Institutes of Health protocol 14-I-0146. Exome-enriched libraries were quantitated using the KAPA library quantification kit and following the manufacturer’s protocol (KAPA Biosystems, Woburn, MA). Library quantifications were size adjusted based on the library peak size estimated from the Bioanalyzer profiles.

Exome-enriched libraries were clustered on the cBot using 10 and 11 pM of template and then sequenced on the Illumina HiSeq 2500 as 2 × 100-bp paired-end reads, following the manufacturer’s protocol (Illumina, San Diego, CA).

Identification of single-nucleotide polymorphisms (SNPs) and indels was performed using Strand next-generation sequencing software. Strand next-generation sequencing utilizes a modified Bayesian variant-calling method adapted from the MAQ SNP-calling algorithm that compares the nucleotides present on aligned reads against the reference at each position in the genome.

CRISPR guide RNAs (gRNAs), used for mouse Stat4 gene E626G knockin, were designed using the CRISPOR Web tool (31). Multiple guides were selected and the one successful for introducing the mutation was 5′-CGACAGTCTCCCTTTGTTGTAGG-3′ (gRNA5) (protospacer adjacent motif [PAM] sequence in green). Synthetic single-guide RNAs were made by Synthego. eSpCas9 recombinant protein was ordered from Millipore (ESPCAS9PRO-250UG). Single-stranded oligodeoxynucleotides with 30- to 70-bp homology to sequences on each side of each gRNA-mediated double-stranded break were designed and ordered from IDT. Silent mutations introducing RsaI restriction site for E626G knockin were created in the corresponding oligonucleotides for genotyping purposes.

Ribonucleoprotein complexes were assembled by incubating recombinant Cas9 protein with single-guide RNA for 10 min at room temperature. Then the single-stranded oligonucleotide was added to the mixture, followed by 10-min centrifugation at 10,000 rpm. The final concentrations used for the microinjections were 50, 30, and 50 ng/μl, respectively.

Fertilized eggs were collected from the oviducts of superovulated BL6/NJ females. Microinjections were performed by continuous flow injection of the ribonucleoprotein/single-stranded oligodeoxynucleotide mixture into the pronucleus of single-cell zygotes.

Tail tipping of the newborn mice was used to purify DNA for genotyping by PCR, employing two screening primers: forward, 5′- CAGACTCCAGAAGGTCAGACG-3′ and reverse, 5′- TCAGAGGGTTTTCAGGGATG-3′, producing a 231-bp band for the WT and two additional bands of 141 and 90 bp in the positive mice when restricted with RsaI.

C. posadasii strain RMSCC1038 (32) (Cp1038) was grown on 2× glucose-yeast extract agar (GYE) (2% glucose, 1% yeast extract, and 1.5% agar) at 30°C for 12 wk by the spin bar method, filtered through Pellon Thermolam Plus to remove hyphal elements, and enumerated by hemocytometer viability, and enumeration was determined by growth of 10-fold serial dilutions on GYE at 37°C for 7 d. Viable suspensions for animal inoculation were diluted in isotonic saline. All manipulation of live fungus was performed at biosafety level 3 with University of Arizona Institutional Biosafety Committee approval.

Six- to 8-wk-old female C57BL/6NJ (stock no. 005304) and C57BL-6J-Stat4em3Adiuj/J (Stat4 knockout, stock no. 028526) mice were purchased from The Jackson Laboratory. B6-Stat4em1Doe/em1Doe (B6-Stat4E626G) mice were maintained in-house as homozygotes. Heterozygous B6-Stat4E626G/+ mice were produced by crossing with C57BL/6NJ mice. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Arizona.

Mice were housed and manipulated at animal biosafety level for all infection and postinfection procedures. Groups of 5–10 mice were infected intranasally under ketamine-xylazine anesthesia with ∼50 coccidioidal spores in 30 µl of isotonic sterile saline. Animals were monitored by observation daily and weekly weight measurement for declining condition and were humanely euthanized when moribund. Mice from fungal burden studies were sacrificed at specified time points and lung and spleen fungal burdens were quantitated by serial dilution of organ homogenates on GYE plates at 35–37°C for up to 7 d as previously described (32). For survival studies, mice were observed for up to 6 wk and moribund mice were sacrificed as needed with quantitative culture of lungs and spleens. For immunologic studies, mice were sacrificed at prespecified time points and lungs, spleens, lymph nodes, and blood were collected for further analysis.

Lungs collected at time points ranging from 2 to 42 d postinfection were split and the right lung was thinly sliced and incubated for 24 h in 2.5 ml of RPMI 1640 medium with 10% FCS at 37°C, 5% CO2 in six-well tissue culture plates (33). Supernatant was centrifuged, filtered through a 0.8-µm filter to sterilize, and stored at −80°C until analysis. Cytokines were measured using a Luminex mouse 31-plex panel (EMD Millipore, Billerica, MA) as previously described. Secretion of single cytokines was confirmed using a DuoSet ELISA (R&D Systems, Minneapolis, MN).

Lungs were perfused with PBS to remove blood and then finely minced. Minced lung was then further processed using a Miltenyi lung dissociation kit and a gentleMACS Octo Dissociator per the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Spleens and lymph nodes were processed by mechanical separation of cells over 70-μm cell strainers as previously described (34). RBCs were lysed by using ammonium chloride–potassium carbonate lysis buffer. Viable cells were enumerated via Vi-Cell (Beckman Coulter, Indianapolis, IN).

The following directly conjugated Abs were used for flow cytometry analysis: B220 clone RA3-6B2, CD3 clone 17A2, CD4 clone GK1.5, CD8 clone 53-6.7, CD11b clone M1/70, CD11c clone N418, CD19 clone 1D3, CD21 clone HB5, CD23 clone B3B4, CD24 clone M1/69, CD25 clone PC61, CD27 clone LG.3A10, CD44 clone IM7, CD45 clone 104, CD62L clone MEL-14, CD49a clone HMα1, CD64 clone 10.1, CD90.2 clone 53-2.1, γδ TCR clone UC7-13D5, IgD clone 11-26c.2a, IgM clone II/41, MHC class II IA/IE clone M5/114.15.2, NK1.1 clone PK136, NKp46 clone 29A1.4, Ly6G clone RB6-8C5, and ST2 clone D1H4. All Abs were titrated on normal B6 splenocytes prior to use. After preparation of single-cell suspensions, cells were blocked for 30 min at 4°C with 24G2 supernatant to block Fc receptors. Cells were then stained with indicated Abs for 30 min at 4°C in the dark. Cells were then washed three times with PBS + 2% BSA and analyzed on a Becton Dickinson LSR II flow cytometer. FlowJo (Tree Star) was used for all flow cytometry analyses. Total cell numbers were calculated based on viable counts.

Coccidioides strain Δcps1 was incubated with mouse sera diluted 1:100 in PBS for 30 min at room temperature. Cells were then washed three times with PBS + 2% BSA. Labeled secondary Abs were incubated for 30 min at room temperature in the dark. Cells were then washed three times with PBS + 2% BSA and analyzed on a Becton Dickinson LSR II flow cytometer. Results are expressed as the change in mean fluorescence intensity compared with preinfection serum.

With regard to clinical presentation, three individuals across three generations within a family all developed DCM. The proband (Fig. 1A) is an African American female who at age 48 y developed pulmonary CM, diagnosed by coccidioidal serology. A concurrent skin lesion was biopsied and showed spherules, verifying extrapulmonary dissemination. Fluconazole treatment resulted in healing of the lesion; however, lesions recurred with fluconazole discontinuation; the patient has continued fluconazole for more than two decades.

FIGURE 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 gRNA, used to generate the E626G mutation in exon 21 of the mouse STAT4 gene. PAM sequence is in green. Oligonucleotide sequence shows the introduced SNPs, one for the intended change E626G, and one to destroy the PAM sequence and prevent recutting, 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.

FIGURE 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 gRNA, used to generate the E626G mutation in exon 21 of the mouse STAT4 gene. PAM sequence is in green. Oligonucleotide sequence shows the introduced SNPs, one for the intended change E626G, and one to destroy the PAM sequence and prevent recutting, 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.

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The proband’s mother, at age 37 y, developed severe coccidioidal pneumonia, which was treated with amphotericin B. A nasal lesion was biopsied and demonstrated spherules, confirming DCM. She completed a course of amphotericin B with resolution of her illness and has not needed further therapy for the past five decades.

The proband’s son developed pulmonary symptoms at age 12 y, suffering a 12-pound weight loss before being diagnosed with CM. He recovered without treatment. At 15 y he suffered a minimal trauma fracture of his left hand. Radiographs demonstrated a lytic lesion in one of the bones, which was subsequently biopsy-proven culture positive to be Coccidioides. He was treated for 1 y on a research protocol with either fluconazole or itraconazole (35), after which no further treatment has been needed for the subsequent 22 y.

Given the dominant inheritance pattern, the three family members underwent whole-exome sequencing. We identified 541 shared coding variants, 30 of which were not present in dbSNPv141. Two of these novel variants, STAT4 c.1877A>G, p.E626G and ALKBH2 c.181C>T, p.R61W, were predicted to be deleterious by multiple algorithms using dbNSFP (36, 37) (Fig. 1B). In silico analysis of the STAT4 variant showed it within the SH2 domain, altering a residue within the highly conserved phospho-tyrosine binding pocket, which is critical for signal transduction. The combined annotation-dependent depletion score for the STAT4 variant was 24.7. At the time of sequencing this mutation was not found in the Genome Aggregation Database (38). Currently there is one variant in Genome Aggregation Database, a frequency of 1/250,864. Given the role of STAT4 in IL-12 signaling and previous demonstration of IL-12RB1 mutations leading to CM, we pursued the STAT4 variant as the causative gene. ALKBH2 is a DNA-repair gene. To date there has been no link between DNA repair and fungal susceptibility; however, a different STAT4 mutation has been implicated in paracoccidioidomycosis (4).

To test the impact of the Stat4E626G mutation in vivo, we used CRISPR/Cas9 to introduce the Stat4E626G mutation into B6/NJ embryos. An ssDNA oligonucleotide was synthesized to drive homology-directed repair to produce the desired substitution based on the published B6 sequence (plus a silent mutation to introduce a convenient restriction site for allele identification) using CRISPR/Cas9 mutagenesis by standard procedures (Fig 1C). (The oligonucleotide and gRNA used are shown in Supplemental Table I.) Embryos were implanted into pseudopregnant females and the resulting pups were genotyped (Fig. 1D). Mutation-positive pups were bred back to WT B6 mice. Heterozygous offspring were then bred to establish a homozygous line.

B6-Stat4em1Doe/em1Doe (noted hereafter as B6-Stat4E626G) mice were fertile and grossly normal. Both heterozygous B6-Stat4E626G/+ and homozygous B6-Stat4E626G/E626G mice showed similar numbers of immune cells and developmental markers compared with WT B6 mice, regardless of genotype (Supplemental Table I). Western blot analysis of spleen cell lysates showed similar levels of STAT4 immunoreactivity in B6, B6-Stat4E626G/+, and B6-Stat4E626G/− mice, whereas B6-Stat4−/− mice had none (Supplemental Fig. 1).

Given the isolated phenotype of DCM in the STAT4(E626G) patients without other notable infections, we sought to determine whether the B6-Stat4E626G mouse recapitulated the patients’ sensitivity to DCM. B6-Stat4E626G mice were intranasally infected with C. posadasii strain RMSCC1038 (Cp1038), which causes lethal infection in B6 mice, typically by 70 d (32). In a study comparing B6 control mice to B6-Stat4E626G heterozygous and homozygous mice, the mice carrying either one or two copies of the mutation had a median survival time (MST) of 38 (p = 0.0072) and 35 d (p = 0.0013), respectively, whereas the control B6 mice survived to the study endpoint day 42 (Fig. 2A).

FIGURE 2.

Mice carrying Stat4 mutations show increased susceptibility to Coccidioides infection.

(A) C57BL/6NJ (black lines), B6-Stat4E626G/+ (red lines), or B6-Stat4E626G/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-Stat4E626G/− (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 three separate experiments of similar design. Significance was determined using a Mantel–Cox log-rank test.

FIGURE 2.

Mice carrying Stat4 mutations show increased susceptibility to Coccidioides infection.

(A) C57BL/6NJ (black lines), B6-Stat4E626G/+ (red lines), or B6-Stat4E626G/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-Stat4E626G/− (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 three separate experiments of similar design. Significance was determined using a Mantel–Cox log-rank test.

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To examine the possibility of STAT4 haploinsufficiency, we crossed WT B6 with B6-Stat4−/− mice, producing Stat4+/− mice, which have only a single functional copy of STAT4. As an additional control, we also bred Stat4E626G/E626G mice with B6-Stat4−/− mice to produce Stat4E626G/− mice. F1 mice along with control Stat4−/− and WT B6 mice were infected with Cp1038 and followed for disease progression. Both Stat4E626G/− mice and Stat4E626G/+ mice succumbed to infection (MST of 35 d). In contrast, B6-Stat4+/− mice with a single WT Stat4 allele were indistinguishable from WT mice (MST >40 d). Therefore, Stat4E626G is dominant negative (Fig. 2B), and Stat4 does not show haploinsufficiency in this challenge.

Because resistance to fatal DCM infection is dependent on CD4+ T cell responses (30, 35, 36), we tested the ability of Stat4E626G T cells to respond to STAT4-dependent signals. To examine whether the induced mutation had an effect on STAT4 signaling, purified splenic CD4+ T cells from naive B6, B6-Stat4E626G/+, or B6-Stat4−/− mice were stimulated with anti-CD3/CD28 beads in the presence of IL-2. After 48 h, media were supplemented with nothing, IL-12, IL-18, or IL-12+IL-18 for an additional 24 h. Supernatants were collected and analyzed for IFN-γ production. Addition of IL-18 or IL-12+IL-18 increased the production of INF-γ in WT-B6 T cells (Fig. 3), whereas B6-Stat4−/− cells produced no IFN-γ under any stimulation. B6-Stat4E626G/+ T cells produced less INF-γ than did WT-B6 cells when stimulated by IL-2+IL-18, IL-2+IL-12+IL-18, or PMA/ionomycin, indicating a deficiency in STAT4 signaling downstream of the IL-12 and IL-18 receptors. Therefore, Stat4E626G protein dominantly inhibits STAT4 function, but does not completely abrogate it (Fig. 3).

FIGURE 3.

CD4+ T cells were purified from C57BL/6NJ (black circles), B6-Stat4E626G/+ (red squares), or B6-Stat4−/− (blue triangles) spleens.

Purified cells were then plated with recombinant mouse IL-2 and activated with CD3/28 DynaBeads according to the manufacturer’s instructions for 48 h. At this point cells were supplemented with the indicated cytokine for 24 more hours. PMA/ionomycin samples were treated for the last 4 h of the experiment. Supernatants were then collected and analyzed for IFN-γ production by ELISA. Graph is representative of two separate experiments of similar design. Significance was determined using a Mann–Whitney test.

FIGURE 3.

CD4+ T cells were purified from C57BL/6NJ (black circles), B6-Stat4E626G/+ (red squares), or B6-Stat4−/− (blue triangles) spleens.

Purified cells were then plated with recombinant mouse IL-2 and activated with CD3/28 DynaBeads according to the manufacturer’s instructions for 48 h. At this point cells were supplemented with the indicated cytokine for 24 more hours. PMA/ionomycin samples were treated for the last 4 h of the experiment. Supernatants were then collected and analyzed for IFN-γ production by ELISA. Graph is representative of two separate experiments of similar design. Significance was determined using a Mann–Whitney test.

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Because STAT4 signaling is depressed and mutant mice have increased mortality to Cp1038, we examined the underlying immune responses. We determined total cellularity in the lungs, mediastinal lymph nodes (msLNs), and spleens of B6-Stat4E626G/+ and WT-B6 mice following Cp1038 infection. Total lung cellularity postinfection increased equivalently in B6-Stat4E626G/+ and WT-B6 mice (Fig. 4A). The draining msLNs also showed increased cellularity during the course of the infection (Fig. 4B). Notably, there was a delayed response in the B6-Stat4E626G/+ apparent at 2 wk postinfection (p = 0.0218), suggesting a delayed adaptive immune response. However, by 4 wk postinfection this difference had normalized. Following this intranasal infection, the spleens showed very little increase in cellularity, indicating that most of the immune reactivity was in the thoracic cavity (Fig. 4C).

FIGURE 4.

Stat4E626G/+ mice have similar cellularity in the lungs and mediastinal lymph nodes after Cp1038 infection.

C57BL/6NJ (black circles), B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (AC) 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 two separate experiments of similar design. Significance was determined using a Mann–Whitney test on log-transformed data.

FIGURE 4.

Stat4E626G/+ mice have similar cellularity in the lungs and mediastinal lymph nodes after Cp1038 infection.

C57BL/6NJ (black circles), B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (AC) 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 two separate experiments of similar design. Significance was determined using a Mann–Whitney test on log-transformed data.

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Because of the decrease in total msLN cells in B6-Stat4E626G mice, the lung cellular composition at 2 wk postinfection was further characterized by multiparameter flow cytometry. The B6-Stat4E626G/+ lungs had a slight increase in total B cells as compared with those from WT-B6 mice (p = 0.0213). This increase was seen in the IgMIgD+ fractions, indicating that these are mature B cells (p = 0.0197) (Fig. 5). Because there was a difference in mature B cells between the mouse strains, we examined serum Abs to Coccidioides. Fluorescence-linked immunosorbent assay examination of serum Abs (IgG1, IgG2B, IgG2C, and IgG3) to Coccidioides showed no differences between the two groups (Supplemental Fig. 2). The T cell populations were not different between B6-Stat4E626G and WT-B6 mice in the lungs (Fig. 5).

FIGURE 5.

Stat4E626G/+ mice have slightly decreased accumulation of B cells the lungs after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A and B) At 2 wk postinfection mice were sacrificed (n = 4–5/group) and total numbers of T (CD3+CD19) (A) and B cells (CD19+B220+) (B) were determined in the lungs by flow cytometry. (CF) B cells were further subtyped by expression of IgM and IgD. Significance was determined by a Mann–Whitney test on log-transformed data. Data are combined from two experiments of similar design.

FIGURE 5.

Stat4E626G/+ mice have slightly decreased accumulation of B cells the lungs after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A and B) At 2 wk postinfection mice were sacrificed (n = 4–5/group) and total numbers of T (CD3+CD19) (A) and B cells (CD19+B220+) (B) were determined in the lungs by flow cytometry. (CF) B cells were further subtyped by expression of IgM and IgD. Significance was determined by a Mann–Whitney test on log-transformed data. Data are combined from two experiments of similar design.

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Given that the msLN cellularity was decreased in B6-Stat4E626G/+ mice 2 wk postinfection, we examined which cells were reduced. There was reduced accumulation of T cells in the msLNs of B6-Stat4E626G/+ compared with WT-B6 mice (Fig. 6A). The defect in cell accumulation was seen in naive, effector, and central memory compartments of both CD4+ and CD8+ T cells (Fig. 6B–G). Additionally, msLNs from B6-Stat4E626G/+ mice failed to accumulate activated B cells as compared with WT-B6 mice (Fig. 7).

FIGURE 6.

Stat4E626G/+ mice have decreased accumulation of total and activated T cells in the mediastinal lymph nodes 2 wk after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. Two weeks postinfection 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 and E) Naive T cells (CD62L+, CD44var). (C and F) Effector T cells (CD62LCD44). (D and G) Memory T cells (CD62LCD44+). Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

FIGURE 6.

Stat4E626G/+ mice have decreased accumulation of total and activated T cells in the mediastinal lymph nodes 2 wk after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. Two weeks postinfection 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 and E) Naive T cells (CD62L+, CD44var). (C and F) Effector T cells (CD62LCD44). (D and G) Memory T cells (CD62LCD44+). Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

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FIGURE 7.

Stat4E626G/+ mice have decreased accumulation of total and activated B cells in the mediastinal lymph nodes 2 wk after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A) At 2 wk postinfection, mice were sacrificed (n = 4–5/time point) and total B cells (CD19+B220+) were enumerated in the mediastinal lymph nodes. (BE) B cells were further phenotyped for their expression of IgM and IgD. Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

FIGURE 7.

Stat4E626G/+ mice have decreased accumulation of total and activated B cells in the mediastinal lymph nodes 2 wk after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A) At 2 wk postinfection, mice were sacrificed (n = 4–5/time point) and total B cells (CD19+B220+) were enumerated in the mediastinal lymph nodes. (BE) B cells were further phenotyped for their expression of IgM and IgD. Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

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To chronicle adaptive immune cell accumulation early in the response, msLNs and lungs were harvested at 2, 9, and 14 d postinfection. At 2 d postinfection there was no increase in lung or msLN cellularity in WT or mutant mice compared with uninfected mice. This lack of very early immune activation is consistent with the fact that Coccidioides spherule rupture with Cp1038 occurs 5 d postinfection (34). However, at 9 d postinfection the msLNs had significantly fewer CD4+ (Fig. 8) and CD8+ (Fig. 9) T cells in the B6-Stat4E626G/+ mice than in the WT-B6 mice, consistent with an impaired adaptive immune response. At 14 d postinfection the T cell numbers were not significantly different in this infection, although the B6-Stat4E626G/+ mice were trending toward lower accumulation, similar to earlier experiments. Additionally we examined the accumulation of innate lymphoid cells (ILCs) in the lung and msLNs on days 2, 7, 10, and 14 postinfection. There were no differences among mouse strains in these cell types in the lungs. In the msLNs, as with other cell types examined, no differences were found between B6 and B6-Stat4E626G/+ mice on day 2 postinfection. There was a decrease in NK cells and the IFN-γ–producing ILC1s between day 7 and 10 in both mouse strains, but the loss was more severe in the B6-Stat4E626G/+ mice. The NK cell and ILC1 numbers recovered in both mouse strains by day 14 (Fig. 10). There were no differences observed in ILC2s and ILC3s.

FIGURE 8.

Stat4E626G/+ mice have decreased accumulation of total and activated CD4+ T cells in the mediastinal lymph nodes early after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A) At the indicated time point postinfection mice were sacrificed (n = 4–5/time point) and total CD4+ T cells were enumerated in the mediastinal lymph nodes. (BD) Cells were further divided into naive (B) (CD62L+, CD44var), effector (C) (CD62LCD44), and memory (D) (CD62LCD44+) subtypes. Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

FIGURE 8.

Stat4E626G/+ mice have decreased accumulation of total and activated CD4+ T cells in the mediastinal lymph nodes early after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A) At the indicated time point postinfection mice were sacrificed (n = 4–5/time point) and total CD4+ T cells were enumerated in the mediastinal lymph nodes. (BD) Cells were further divided into naive (B) (CD62L+, CD44var), effector (C) (CD62LCD44), and memory (D) (CD62LCD44+) subtypes. Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

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FIGURE 9.

Stat4E626G/+ mice have decreased accumulation of total and activated CD8+ T cells in the mediastinal lymph nodes early after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A) At the indicated time point postinfection mice were sacrificed (n = 4–5/time point) and total CD8+ cells were enumerated in the mediastinal lymph nodes. (BD) Cells were further divided into naive (B) (CD62L+, CD44var), effector (C) (CD62LCD44), and memory (D) (CD62LCD44+) subtypes. Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

FIGURE 9.

Stat4E626G/+ mice have decreased accumulation of total and activated CD8+ T cells in the mediastinal lymph nodes early after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A) At the indicated time point postinfection mice were sacrificed (n = 4–5/time point) and total CD8+ cells were enumerated in the mediastinal lymph nodes. (BD) Cells were further divided into naive (B) (CD62L+, CD44var), effector (C) (CD62LCD44), and memory (D) (CD62LCD44+) subtypes. Significance was determined by a Mann–Whitney test of log-transformed data. Data are combined from two experiments of similar design.

Close modal
FIGURE 10.

Stat4E626G/+ mice have decreased accumulation of NK cells and ILC1s in the mediastinal lymph nodes early after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time point postinfection mice were sacrificed (n = 3–5/time point) and total NK cells (NK1.1+, NKp46+, CD49a) and ILC1s (NK1.1+, NKp46+, CD49a+) were enumerated in the mediastinal lymph nodes. Significance was determined by a Mann–Whitney test of log-transformed data.

FIGURE 10.

Stat4E626G/+ mice have decreased accumulation of NK cells and ILC1s in the mediastinal lymph nodes early after Cp1038 infection.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time point postinfection mice were sacrificed (n = 3–5/time point) and total NK cells (NK1.1+, NKp46+, CD49a) and ILC1s (NK1.1+, NKp46+, CD49a+) were enumerated in the mediastinal lymph nodes. Significance was determined by a Mann–Whitney test of log-transformed data.

Close modal

Innate cell recruitment to the lungs was not different between the WT and mutant mouse strains, indicating that Stat4E626G mutation did not affect initial recognition of Coccidioides, innate recruitment, or retention (Supplemental Fig. 3).

IFN-γ responses are crucial to recovery from CM in humans and mice (37). As expected, STAT4 is also critical as it functions in both CD4 and CD8 T cells as an regulator of the IFN-γ production. We hypothesized that the decreased STAT4 signaling observed in vitro would also result in lowered IFN-γ responses in vivo. To test this, we characterized the differences in secretion of cytokines following CM in B6-Stat4E626G and WT-B6 mice infected intranasally with Cp1038 and sacrificed 2, 4, 5, and 6 wk later. Lungs were collected to determine both cellular composition and cytokine production. Spleens, peripheral blood, and msLNs were analyzed for total cell number and cellular composition. Both B6-Stat4E626G and WT-B6 mice showed similar increases in lung weights, a general measure of disease progression (39), beginning at 4 wk postinfection (Supplemental Fig. 3). Lung fungal burdens were similar in both B6-Stat4E626G and WT-B6 mice, except at 5 wk postinfection, when they were greater in B6-Stat4E626G/+ mice (Fig. 11A). Splenic fungal burden was absent at 2 wk postinfection but identical thereafter in both B6-Stat4E626G and WT-B6 mice (Fig. 11B).

FIGURE 11.

Stat4E626G/+ mice have similar fungal burdens after Cp1038 infection as compared with B6 mice.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A and B) At the indicated time point postinfection mice were sacrificed (n = 4–5/time point) and fungal burdens were determined in the lungs (A) and spleen (B). Significance was determined by a Mann–Whitney test of log-transformed data. Data are representative of two experiments of similar design.

FIGURE 11.

Stat4E626G/+ mice have similar fungal burdens after Cp1038 infection as compared with B6 mice.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. (A and B) At the indicated time point postinfection mice were sacrificed (n = 4–5/time point) and fungal burdens were determined in the lungs (A) and spleen (B). Significance was determined by a Mann–Whitney test of log-transformed data. Data are representative of two experiments of similar design.

Close modal

To examine the cytokine milieu in the lungs directly, thinly sliced lung sections were cultured overnight and cytokine secretion was measured in the supernatants by Luminex. Differences in cytokine levels were reanalyzed by targeted ELISA to confirm the results of the screening assay. Lung slices from WT-B6 mice demonstrated upregulation of IFN-γ (Fig. 12A) and IFN-γ–related cytokines IP-10 (Fig. 12B) and MIG (Fig. 12C) postinfection. In contrast, B6-Stat4E626G/+ mice failed to increase production of these IFN-γ–dependent cytokines and chemokines. These in vitro results correlated with serum cytokine levels (Fig. 12D–F).

FIGURE 12.

Stat4E626G/+ mice have decreased production of IFN-γ and related cytokines after Cp1038 infection as compared with B6 mice.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time point postinfection mice were sacrificed (n = 4–5/time point). Lungs were finely sliced and placed in complete RPMI for 24 h. (AC) Supernatant was then collected and analyzed for IFN-γ (A), IP-10 (B), and MIG (C) cytokine production by ELISA. (DF) Serum was harvested and analyzed for IFN-γ (D), IP-10 (E), and MIG (F) by ELISA. Significance was determined by a Mann–Whitney test.

FIGURE 12.

Stat4E626G/+ mice have decreased production of IFN-γ and related cytokines after Cp1038 infection as compared with B6 mice.

C57BL/6NJ (black circles) and B6-Stat4E626G/+ (red squares) mice were intranasally infected with Cp1038. At the indicated time point postinfection mice were sacrificed (n = 4–5/time point). Lungs were finely sliced and placed in complete RPMI for 24 h. (AC) Supernatant was then collected and analyzed for IFN-γ (A), IP-10 (B), and MIG (C) cytokine production by ELISA. (DF) Serum was harvested and analyzed for IFN-γ (D), IP-10 (E), and MIG (F) by ELISA. Significance was determined by a Mann–Whitney test.

Close modal

These results demonstrate a dominant negative effect of STAT4E626G on the function of STAT4 both in vivo and in vitro in mice, modeling the effect in humans. Mice carrying the mutation associated with human DCM had significantly more rapid disease progression than did WT mice. Similarly, the apparently normal lymphoid system of the E626 mice is similar to the apparently normal immune system in the patients.

We identified a multigenerational family with DCM associated with a novel heterozygous mutation, STAT4 E626G. As with the patients, mice heterozygous for Stat4E626G were more susceptible to coccidioidal infection. These mice showed normal development of immune cells in both primary and secondary lymphoid compartments, indicating that this mutation did not lead to a global defect in immune system development. This is not surprising because Stat4 knockout mice are also grossly normal (2, 40).

STAT4 regulates the IL-12/IFN-γ pathway in both humans and mice. A variety of mutations in the IL-12/IFN-γ pathways have been described predisposing to a variety of fungal diseases (28, 4143). Although a complete loss of STAT4 function in knockout mice dramatically ablates the IFN-γ response and results in the loss of Th1 function, little information is available on specific missense mutations. STAT4 E651V mutation was reported to reduce in vitro STAT4 phosphorylation and nuclear translocation in a family with Paracoccidioides brasiliensis (4). Although Stat4E626G is expressed at levels similar to WT Stat4, CD4+ T cells were deficient in making IFN-γ in response to IL-12+IL-18. Mice either heterozygous or homozygous for Stat4E626G were similarly susceptible to infection with Coccidioides, indicating that one copy of mutant Stat4 was sufficient for infection sensitivity whereas mice heterozygous for a null allele (Stat4+/−) were phenotypically WT, confirming Stat4E626G as a dominant negative allele.

The immune response after intranasal Coccidioides infection of B6-Stat4E626G mice showed relatively few global differences compared with those in B6 controls. Spleens showed no increase in total cellularity, indicating that most of the immune response was limited to the lungs and draining msLNs. The cellular accumulation into the lungs showed few differences. The proliferation and homing of T cells was unaffected, although B6-Stat4E626G/+ mice did have a slightly elevated number of B cells compared with controls. In contrast to the relatively normal cell numbers, supernatants from lung homogenates from infected B6-Stat4E626G/+ mice made significantly less IFN-γ and IFN-γ–driven chemokines than did controls. The IFN-γ that was detected 4–5 wk postinfection was most likely produced as a result of adaptive immune responses. These defects in IFN-γ and its downstream-induced genes are overall consistent with impaired expression of T-bet and reduced polarization of CD4 cells toward Th1 responses, as well as IFN-γ production of CD8 T cells.

Many groups have shown a dependence of IFN-γ for both survival of Coccidioides infection as well as development of protective immunity by experimental vaccination (30, 4447). Of note, there was no detectable IL-12p40 or IL-12p70 in the lung supernatants of either WT-B6 or B6-Stat4E626G mice, consistent with these low-abundance, IFN-γ–inducing cytokines being consumed in a paracrine manner and not detectable in culture supernatants.

The most significant alterations in the immune responses were found in the draining msLNs. B6-Stat4E626G/+ mice failed to accumulate activated T cells and B cells during the early phase of infection, <2 wk postinfection. At 2 d postinfection, before the first rupture of Coccidioides endospores, there was no increase in cellularity in the msLNs in either WT-B6 or B6-Stat4E626G mice. This indicates little widespread immune response or activation to the initial infection, although it is possible that there may have been some small local responses in the lung. At 9 and 14 d postinfection, B6-Stat4E626G/+ mice had significantly fewer cells in their msLNs. At 2–6 wk post infection, although there was no difference between WT-B6 or B6-Stat4E626G total lung cellularity, there was significant and persistent reduction in cellularity in the msLNs. Whether this cellular accumulation is recruitment from the periphery or local expansion remains to be explored. While this defect in cellularity is subtle, it has a profound effect on disease severity and time to death. Interestingly, the fungal burdens in lungs and spleen as well as lung weights were similar between WT-B6 or B6-Stat4E626G mice. Therefore, the early death seen in B6-Stat4E626G mice was not due to fungal proliferation or lung vascular permeability but correlated most closely with the impaired IFN-γ responses. Importantly, note that STAT4 is also involved in IL-23 signaling, another cytokine critically important to mycobacterial and fungal responses (4852). Improperly high IL-4 production has been seen in other fungal infections, including Histoplasma, Aspergillus, and Candida (5355), and we detected elevated IL-4 in the serum and lungs of some of infected B6-Stat4E626G/+ mice, but as a group the difference was not significantly different from B6 mice. Investigation of the effective cause of early mortality in the B6-Stat4E626G mice is ongoing.

Dominant negative mutation in STAT4 impairs the production of IFN-γ and its downstream effectors. It is noteworthy that our patients were apparently not subject to other severe viral, bacterial, or fungal infections, suggesting that STAT4 is redundant for most immune responses required in everyday life. However, this mutation predisposed them, so far apparently uniquely, to DCM, highlighting the importance of host genetics in human coccidioidal infections.

This work was supported by a grant from the National Institutes of Health (U01AI122275) to J.N.G. and S.M.H.

D.A.P., S.M.H., J.N.G., and J.A.F. designed the study methodology. D.A.P., A.P.H., L.F.S., C.D.B., H.M., H.T.T., M.J.O., D.M.R., and J.E.W. collected specimens and experimental data. T.D. and T.G.G. generated and validated the model mouse. D.A.P., A.P.H., S.M.H., J.N.G., and J.A.F. analyzed data. D.A.P. wrote the original draft of the manuscript. All authors reviewed and edited the manuscript.

The online version of this article contains supplemental material.

Abbreviations used in this article

CM

coccidioidomycosis

Cp1038

C. posadasii strain RMSCC1038

DCM

disseminated CM

gRNA

guide RNA

GYE

glucose-yeast extract agar

ILC

innate lymphoid cell

msLN

mediastinal lymph node

MST

median survival time

PAM

protospacer adjacent motif

SNP

single-nucleotide polymorphism

WT

wild-type

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The authors have no financial conflicts of interest.

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