Conceived and designed the experiments: DEB MWM MCP RNN CSD. Performed the experiments: DEB MWM MCP RNN CSD. Analyzed the data: DEB MWM MCP RNN CSD. Wrote the paper: DEB CSD.
Current address: Supergen, Salt Lake City, Utah, United States of America
The authors have declared that no competing interests exist.
Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory clinical syndrome associated with neoplastic disorders especially lymphoma, autoimmune conditions, and infectious agents including bacteria, viruses, protozoa and fungi. In both human and veterinary medicine, hemophagocytic histiocytic disorders are clinically important and frequently fatal. HLH in humans can be a primary (familial, autosomal recessive) or secondary (acquired) condition, with both types generally precipitated by an infectious agent. Previously, no mouse model for secondary HLH has been reported. Using
Hemophagocytic lymphohistiocytosis (HLH), an inflammatory syndrome characterized by over-activation of macrophages and T lymphocytes, can be triggered by diverse eukaryotic, bacterial (especially intracellular), and viral pathogens
HLH can be primary (familial, fHLH) or secondary (sHLH). Familial HLH is autosomal recessive, typically diagnosed in infants or children, fatal if untreated
Secondary HLH occurs in all age groups and is associated with infections across classes, malignancies especially lymphoma
Data herein demonstrate that
One-week PI | Three-weeks PI | Six-weeks PI | Ten-weeks PI | |
5/6 mice | 4/6 mice | 6/7 mice | 2/2 mice | |
Days 17–23 |
||||
2X |
3X (1-14X)5/6 mice; 10/10 mice |
3X |
3X (1-3X)1/2 mice | |
2/6 mice(7.7–11.1)1/6 mice |
5/6 mice |
3/6 mice |
0/2 mice(10.6–10.7)0/2 mice(51–55) | |
0/6 mice | 2/6 mice(100) | 2/6 mice(100) | 0/6 mice | |
No | 2/6 mice;3/10 mice |
No | No | |
Range CFU/g: spleen | 102–104 | 104–105 | 103–104 | 101–102 |
liver | 102–104 | 103–104 | 102–103 | None detected |
7/21 mice- onset Day 5–12 | 6/21 mice – signs gone by Day 18 | 1/21 mice- early death Day 37 | None | |
ΠChronic active hepatitis | Acute hepatitis 5/6 mice | Chronic active hepatitis 5/6 mice | Chronic active hepatitis 7/7 mice | Chronic active hepatitis 1/2 mice |
PI indicates post-oral infection with 2.0×109 CFU
Independent experiment, same bacterial dosing and range for splenic bacterial CFU results.
Formal diagnostic criteria for HLH per the Histiocyte Society guidelines
Consistent with a diagnosis of HLH, and Π strong supportive evidence for HLH
Our findings of neurological disease, splenomegaly and inflammatory lesions are consistent with prior descriptions of murine
Mice were orally gavaged with 9.1×108 CFU of
Parameter | Week 1 | Week 3 | Week 6 | Week 10 | ||||
C, n = 4 | I, n = 6 | C, n = 4 | I, n = 6 | C, n = 3 | I, n = 6 | C, n = 1 | I, n = 2 | |
WBC×103/µl | 5.73 |
5.32 |
7.23 |
5.32 |
7.03 |
8.13 |
6.8 | 14.8, 6.7 |
RBC×106/µl | 9.79 |
9.70 |
9.79 |
9.20 |
9.64 |
11.42 |
8.86 | 11.43, 9.42 |
Neutrophils x103/µl | 0.50 |
1.35 |
0.75 |
1.25 |
0.63 |
1.82 |
0.40 | 4.00, 0.60 |
Monocytes x103/µl | 0.07 |
0.17 |
0.10 |
0.23 |
0.10 |
0.43 |
0.10 | 0.50, 0.10 |
Lymphocytes x103/µl | 4.73 |
3.57 |
6.28 |
3.72 |
6.00 |
5.70 |
6.10 | 10.10, 5.80 |
HCT, % | 53 |
51 |
53 |
46 |
53 |
54 |
51 | 55, 51 |
MCV, fl | 54.0 |
52.0 |
54.3 |
49.7 |
55.7 |
47.3 |
58.0 | 48.0, 54.0 |
MCVr, fl | 59.3 |
56.3 |
59.3 |
52.8 |
62.0 |
55.8 |
62.0 | 59.0, 63.0 |
MCHC, g/dl | 31.0 |
31.5 |
31.3 |
30.0 |
30.0 |
30.0 |
30.0 | 30.0, 31.0 |
RDW | 12.4 |
12.7 |
12.5 |
16.7 |
12.7 |
14.6 |
12.6 | 14.3, 13.0 |
Platelets x106/µl | 1.02 |
1.00 |
0.92 |
0.54 |
1.05 |
0.79 |
0.87 | 1.06, 1.07 |
MPV, fl | 7.3 |
7.1 |
7.3 |
7.2 |
7.7 |
8.2 |
6.7 | 7.1, 6.7 |
Reticulocytes x106/µl | 0.48 |
0.41 |
0.40 |
0.71 |
0.44 |
0.41 |
0.57 | 0.54, 0.64 |
CHr, pg | 17.9 |
16.7 |
17.7 |
15.0 |
17.4 |
15.5 |
17.4 | 15.817.8 |
Serum Iron µg/dl | 262 |
297 |
267 |
219 |
269 |
266 |
222 | 202, 280 |
Twenty-one mice were orally gavaged with 2.0×109 CFU of
WBC indicates white blood cells; RBC, red blood cells; HCT, hematocrit; MCV, mean cell volume; MCVr, mean cell volume reticulocytes; MCHC, mean cell hemoglobin concentration; RDW, red cell distribution width; MPV, mean platelet volume; and CHr, mean cell hemoglobin reticulocytes.
Infected mice developed slight fevers and clinical neurological signs including ataxia, head tilt, uni-directional circling when ambulating, and uni-directional rotatory spinning when lifted by the tail in seven of twenty-one mice (
Hematological findings included acute and ongoing response to infection characterized by immediate onset of neutrophilia (
Infected mice had early onset slight to moderate microcytic anemia which was most severe at three weeks post-infection, correlating with highest bacterial tissue loads and most severe splenomegaly (
(A) Control bone marrow cytology, 3 weeks post mock-infection. (B) Infected mouse bone marrow cytology, 3 weeks post-infection; myeloid hyperplasia with increased blasts and monocytes, hemophagocytic macrophage (white arrow), and foamy macrophage (black arrow). (C) Bone marrow histology, 6 weeks post-infection; hypercellularity with myeloid hyperplasia. (D) Mouse bone marrow cytology, 3 weeks post-infection; erythrocyte (arrow and inset) within hemophagocytic macrophage. (E) Control mouse blood film, 3 weeks post mock-infection. (F) Blood film from highly infected mouse, 3 weeks post-infection; marked erythrocyte anisocytosis, increased polychromasia and markedly fragmented erythrocytes (mature and polychromatophilic). Polychromatophilic erythrocytes (arrow-heads), fragmented polychromatophilic erythrocytes (carats), fragmented erythrocytes (arrows), L = small lymphocyte. Wright stain (A, B, D, E, F). H & E stain (C). Original magnifications 500× (A–B, D), 200× (C), or 1000× (E–F).
Thrombocytopenia present from weeks 1–10 (
Serum IgG titers to
Splenomegaly was present in six of seven infected mice at the conclusion of the 16-week study (
Bone marrows were hypercellular (
(A) Mouse liver, 6 weeks post-infection; inflammation and necrosis (arrow). (B) Mouse spleen, 3 weeks post-infection; extramedullary hematopoiesis (EMH; arrow, megakaryocytes), histiocytic infiltration (I) throughout the red pulp, and thrombus (T). H&E stain (A, B). (C) Spleen, mock-infected (left) and infected mouse (right), 3 weeks post-infection; markedly decreased ferric iron staining in red pulp. (D) Spleen, mock-infected (left) and infected mouse (right), 6 weeks post-infection; markedly decreased splenic ferric iron in red pulp. Perl's Prussian Blue stain (C, D). (E) Hemophagocytic macrophage in mouse spleen 3 weeks post-infection that had 10-fold more macrophages and 43-fold more 6N+ macrophages than control mouse spleen. CD11b (red), DAPI (blue), TER119 (green). N = endogenous macrophage nucleus, E1 = nucleated erythrocyte, E2 = non-nucleated erythrocyte. Confocal fluorescent micrograph. (F) Representative histogram overlay of TER119 expression on DAPI+ splenocytes from a mock-infected (red) and infected mouse (blue) 3 weeks post-infection. Filled gray histogram corresponds to the isotype control. The infected mouse had 11.5-fold more TER119med pro-erythroblasts and 5.5-fold more TER119high erythroblasts than the mock-infected mouse. (G) Mean numbers of TER119med and TER119high splenocytes from three mock-infected (white bars) and four infected (gray bars) mice. Mean number of TER119med pro-erythroblasts per spleen increased 6.8-fold in infected mice, while the mean number of TER119high cells, corresponding to all nucleated erythroblasts subsequent to the pro-erythroblast stage
To further evaluate erythropoiesis, enlarged spleens from Salmonella-infected mice were analyzed by flow cytometry three weeks after infection. Results from three replicate experiments showed increased numbers of erythroblasts compared to controls as measured by TER119 staining
Increased numbers of hemophagocytic macrophages in the bone marrow or spleen are consistent with HLH
Tissue inflammation and thrombosis occurred in
Serum iron was decreased at one (two of six mice) and three (four of six mice) weeks (
To summarize results, evaluations of blood, bone marrow, liver and spleen of
Infection of Sv129S6 wild-type mice with
While a diagnosis of HLH cannot be solely based on finding hemophagocytic macrophages, an increased number especially in bone marrow cytology (
Activated macrophages are integral to the development of HLH
The pathogenesis of bi-cytopenia in this mouse model is likely multifactorial. Adequate to increased megakaryocytes in bone marrow (
The mouse strain chosen for this study was important given the effect of murine genetic variability on iron homeostasis
Due to expansion of red pulp by EMH and inflammation, splenomegaly persists in mice even after bacterial CFU have cleared. Persistent splenomegaly also occurs in human HLH
Approximately 30% of the mice in our studies developed clinical neurological signs from which most recovered. The incidence of neurological disease in HLH patients varies from 37% in children
Abnormal lipid processing is known to occur with HLH
The important findings in highly infected mice in this chronic systemic infection model are those demonstrating first, an HLH syndrome and second, anemia different from ACD and acute murine
Research protocols were in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and approved by the University of Colorado Institutional Biosafety and Animal Care and Use Committees.
To characterize hematological responses and establish clinico-pathologic features of HLH in
Freshly struck colonies of virulent
Blood (50–150 µL) was collected bi-weekly by retro-orbital method into Microvette K2-EDTA tubes (Sarstedt, Inc; Newton, NC) for CBC using a Hemavet HV950FS analyzer (Drew Scientific; Waterbury, CT). Simultaneously, serum was collected from one mock-infected control and five infected mice for IgG to
The submandibular vascular bundle collection method replaced retro-orbital bleeding to minimize distress to the animal and allow for greater collection volume
Following euthanasia whole spleen and liver were weighed; a section of each was weighed, homogenized and plated for bacterial CFU. No CFU were found in any mock-infected animals. Remaining liver and spleen, and one femur were collected into 10% neutral buffered formalin for histological evaluation. Bone marrow brush smear preparations were made immediately post-mortem from the second femur, and Wright-stained for cytological examination. Tissues were processed by routine histological methods; paraffin sections were stained with hematoxylin and eosin (H & E), Perl's Prussian blue for ferric iron (hemosiderin), and Masson's trichrome stain for fibrosis
Ferritin was quantified using a commercially available mouse ELISA kit (E-90F, Immunology Consultants Laboratory, Inc; Newberg, OR). Absorbance at 450 nm was determined with a plate reader; ferritin concentrations were calculated based on standard curves developed within the same plate and corrected for sample dilution. To verify findings, serum ferritin was measured in duplicated infection experiments using an additional 10
Spleens from mice collected three weeks after
Mann-Whitney U Test and Student's
The authors acknowledge Elizabeth Chlipala, Premier Laboratory, for expert advice and use of the Hemavet 950 analyzer; Dr. Linda Vap and Staff, Colorado State University, for expert advice and use of the Advia 120 hematology analyzer; Todd Bass and Staff, Colorado State University, for expert advice and histochemistry.