Cytokine modulation correlates with severity of monkeypox disease in humans.

BACKGROUND: Human monkeypox is a zoonotic disease endemic to parts of Africa. Similar to other orthopoxviruses, virus and host have considerable interactions through immunomodulation. These interactions likely drive the establishment of a productive infection and disease progression, resulting in the range of disease presentations and case fatality rates observed for members of the Orthopoxvirus genus.
OBJECTIVES: Much of our understanding about the immune response to orthopoxvirus infection comes from either in vitro or in vivo studies performed in small animals or non-human primates. Here, we conducted a detailed assessment of cytokine responses to monkeypox virus using serum from acutely ill humans collected during monkeypox active disease surveillance (2005-2007) in the Democratic Republic of the Congo. STUDY
DESIGN: Nineteen serum samples that were from patients with confirmed monkeypox virus infections were selected for cytokine profiling. Cytokine profiling was performed on the Bio-Rad Bioplex 100 system using a 30-plex human cytokine panel.
RESULTS: Cytokine profiling revealed elevated cytokine concentrations in all samples. Overproduction of certain cytokines (interleukin [IL]-2R, IL-10, and granulocyte macrophage-colony stimulating factor were observed in patients with serious disease (defined as >250 lesions based on the World Health Organization scoring system).
CONCLUSIONS: The data suggest that cytokine modulation affects monkeypox disease severity in humans.

Background

Monkeypox virus (MPXV) is an orthopoxvirus similar to smallpox, and can cause an acute systemic lesional disease associated with high morbidity. Over the last 30 years there has been a marked increase in the number of cases reported in the Democratic Republic of the Congo (DRC) [1].

Immunopathogenesis is believed to play a major role in disease severity and outcome during orthopoxvirus infections. Cytokine storm during variola virus infection has been suggested in human cases and non-human primate models [2], [3], [4]. Induction of a cytokine storm is driven by inflammatory mediators and cellular constituents which lead to overproduction of inflammatory cytokines, resulting in massive inflammation, sepsis and septic shock [5]. To date, the presence of cytokine storm following MPXV infection is predicted but yet to be empirically demonstrated.

Objectives

To determine if a cytokine storm is associated with MPXV infection, serum samples were collected from individuals presenting with febrile illness and rash during active monkeypox disease surveillance in the DRC (2005–2007). Monkeypox infection was confirmed by polymerase chain reaction (PCR) [1].

Study design

Sample acquisition and processing

Methods for sample acquisition and processing are published elsewhere [1]. In MPXV-infected patients [1], disease severity (mild or <25 lesions, moderate or 25–99 lesions, severe or 100–250 lesions, and serious or >250 lesions) was determined using the World Health Organization scoring system used during smallpox eradication [6], [7]. Samples were stratified based on geographic location, disease severity, and transmission source (human or animal). Nineteen cases were selected for cytokine profiling. The breakdown of disease severity was: mild = 4 cases; moderate = 5 cases; severe = 5 cases; serious = 5 cases. Ethical approval was obtained from participating institutions, and informed consent was obtained from patients and parents/guardians.

Human cytokine analysis

Serum cytokines were measured in triplicate using Cytokine Human Magnetic 30-Plex Panel (Life Technologies, Grand Island, NY). Briefly, serum samples were thawed, vortexed, and centrifuged to remove any cryoprecipitants. Pre-mixed, lyophilized stock cytokines were rehydrated and serially diluted 3-fold to produce the standard curve. Fifty microliters of standards, or blanks were assayed per replicate well as per manufacturer's instructions (Life Technologies, Grand Island, NY). Data were acquired on a Bio-Rad Bioplex 100 system (Bio-Rad Laboratories, Hercules, CA), and exported into GraphPad Prism (GraphPad Software, La Jolla, CA) for analysis. Normal human cytokine ranges and averages were obtained from the Bio-Plex Suspension Array System Technical Note 6029 available on the Bio-Rad website (www.bio-rad.com). Statistical significance was determined using unpaired T tests conducted at the 95% confidence level.

Results

Cytokine responses are predictive of disease severity

Nineteen serum samples (Table 1 ) from confirmed MPXV infections were analyzed using a multiplex cytokine assay. Although concentrations of interleukin (IL)-2R were similar between mild, moderate, and severe cases, concentrations were significantly higher (P  < 0.05) for serious cases (Table 2 ). MIP-1α and MIP-1β concentrations in all cases were elevated, and concentrations of these cytokines were significantly elevated (P  < 0.05) in mild cases compared to moderate and severe cases. Although all cases had elevations in serum concentrations of IL-1RA, IL-6, and IL-15, moderate cases had significantly lower concentrations of IL-1RA (this difference was not statistically significant [P  > 0.05]), severe cases had significantly lower concentrations of IL-6 (P  < 0.05), and moderate and severe cases had lower concentrations of IL-15 compared to other disease categories (this difference was not statistically significant [P  > 0.05]). IL-10 concentrations were elevated above normal range for all categories of disease severity; however, the difference between serious cases and cases in all other severity categories was statistically significant (P  < 0.05). IL-10 concentrations were roughly proportional to disease severity. Differences between mild, moderate, and severe were not statistically significant (P  > 0.05). Granulocyte macrophage-colony stimulating factor (GM-CSF) was noticeably elevated above normal human range only for serious disease cases; concentrations in mild, moderate, and severe disease were not significantly different (P  > 0.05).

Table 1

Metadata of 19 patients infected with monkeypox from health zones within the Sankuru District of the Democratic Republic of the Congo.

Patient ID	Health zone	Age	Sex	Disease severitya	Infection source	Monkeypox lineage	Genbank accession #
06MPX0966	Katako Kombe	20.0	M	Mild	Unknown	A	–
06MPX0970	Katako Kombe	10.0	F	Serious	Human	C	JX878408
06MPX1082	Katako Kombe	24.0	M	Severe	Human	ND	–
06MPX1092	Kole	10.0	F	Mild	Animal	A	–
06MPX1094	Kole	1.5	F	Serious	Human	ND	–
07MPX0013	Kole	24.0	F	Mild	Human	B	–
07MPX0016	Kole	2.6	F	Moderate	Human	B	–
07MPX0019	Katako Kombe	9.0	F	Severe	Human	ND	–
07MPX0021	Katako Kombe	13.0	F	Severe	Human	A	–
07MPX0104	Bena Dibele	4.0	F	Serious	Unknown	D	JX878417
07MPX0109	Lodja	3.0	M	Serious	Human	ND	–
07MPX0275	Djalo Ndjeka	10.0	F	Moderate	Human	C	JX878419
07MPX0281	Djalo Ndjeka	17.0	M	Moderate	Animal	C	–
07MPX0286	Lomela	20.0	M	Serious	Animal	A	JX878421
07MPX0412	Katako Kombe	3.8	F	Moderate	Unknown	C	–
07MPX0435	Djalo Ndjeka	1.8	M	Severe	Human	ND	–
07MPX0438	Djalo Ndjeka	10.0	F	Mild	Human	ND	–
07MPX0450	Kole	11.0	M	Severe	Human	C	JX878426
07MPX0496	Katako Kombe	29.0	F	Moderate	Human	C	–

ND = not determined.

Disease severity based on the World Health Organization lesion scoring system.

Table 2

Serum concentrations of cytokines/chemokines in patients with monkeypox infection.

Serum cytokine/chemokine/growth factor concentrations (pg/ml)
Cytokines, chemokines, or growth factors	Normal rangea	Mild diseaseb (SEM)	Moderate diseaseb (SEM)	Severe diseaseb (SEM)	Serious diseaseb (SEM)
IL-1β	0.02–0.70	134.77 (43.28)	649.17 (314.10)	1002.01 (468.50)	144.82 (67.79)
IL-1RA	0.20–665.00	7586.08 (2046.00)	2656.27 (502.60)	7480.04 (2946.00)	6983.32 (2108.00)
IL-2	0.03–90.00	36.08 (9.19)	4.31 (1.97)	55.42 (27.49)	71.02 (37.18)
IL-2R	28.00–594.00	1327.69 (186.70)	1806.10 (154.80)	1557.05 (168.80)	4239.94 (401.60)c
IL-4	0.01–3.00	170.94 (40.59)	298.23 (8.44)	206.17 (28.35)	258.27 (27.28)
IL-5	0.01–7.00	13.25 (1.02)	15.07 (0.61)	16.12 (0.44)	13.75 (0.50)
IL-6	0.02–9.00	314.09 (84.87)	795.90 (374.30)	23.12 (7.10)c	200.82 (48.67)
IL-7	0.01–14.00	25.88 (8.45)	0.00 (0.00)	8.63 (3.79)	8.45 (4.55)
IL-8	0.08–116.00	13,934.80 (2767.00)	11,242.30 (3892.00)	10,232.60 (3613.00)	7944.93 (2736.00)
IL-10	5.90–637.00	5.07 (1.39)	9.23 (1.04)	24.54 (9.54)	377.05 (171.60)c
IL-12p40	36.00–646.00	562.22 (116.10)	692.93 (16.98)	512.59 (70.32)	798.86 (90.67)
IL-13	0.01–9.00	24.83 (3.41)	25.64 (3.33)	33.24 (2.73)	26.05 (3.42)
IL-15	0.06–5.00	198.03 (83.50)	32.31 (15.93)	53.08 (10.18)	190.49 (95.07)
IL-17	0.22–31.00	97.40 (19.42)	100.01 (6.61)	93.12 (8.76)	89.04 (9.79)
IFN-α	3.30–63.00	87.06 (17.14)	55.80 (4.96)	54.12 (4.65)	60.09 (7.29)
IFN-γ	0.60–124.00	86.42 (21.16)	145.54 (5.20)	112.31 (13.96)	135.50 (15.28)
TNF-α	0.10–98.00	16.03 (3.71)	35.62 (8.12)	17.41 (1.93)	22.15 (2.96)
GM-CSF	0.80–122.00	8.86 (3.41)	89.62 (44.43)	101.71 (32.39)	839.00 (421.90)c
Eotaxin	1.20–39.00	54.00 (11.51)	37.35 (4.41)	24.05 (4.43)	72.93 (23.15)
MCP-1/CCL2	2.00–48.00	4128.88 (751.00)	6253.45 (2461.00)	1514.07 (338.60)	3621.06 (966.10)
MIP-1α/CCL3	0.01–2.00	480.30 (107.90)c	170.10 (7.51)	188.17 (26.48)	323.10 (88.28)
MIP-1β/CCL4	1.70–47.00	899.01 (240.70)c	167.85 (20.04)	207.37 (20.17)	354.69 (95.02)
RANTES/CCL5	100.00–2282.00	22,197.50 (10,134.00)	48,143.00 (12,034.00)	28,224.70 (9701.00)	101,828.00 (41,612.00)
MIG/CXCL9	86.00–7911.00	557.48 (108.40)	370.46 (39.88)	403.00 (90.98)	1887.37 (546.70)
IP-10/CXCL10	5.90–637	61.23 (15.69)	80.05 (13.52)	66.78 (16.33)	525.07 (167.00)

Abbreviations: SEM, standard error of the mean; TNF-α, tumor necrosis factor-alpha; IFN, interferon; IL, interleukin; IL-1RA, interleukin-1 receptor antagonist; IL-2R, interleukin-2 receptor; IL-12p40, interleukin-12 p40 subunit; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1alpha; RANTES, regulated upon activation normal T cell expressed and presumably secreted; GM-CSF, granulocyte macrophage colony-stimulating factor; SEM, standard error of the mean.

Values were obtained from Technical Note 6029 “Bio-Plex suspension array system” from Bio-Rad (www.bio-rad.com).

Values represent the average for samples belonging to the specific disease category (mild, moderate, severe, serious).

P < 0.05 compared to other severity categories.

Serum concentrations of IL-1β, IL-1RA, IL-2R, IL-4, IL-5, IL-6, IL-8, IL-13, IL-15, IL-17, MCP-1, and RANTES were consistently elevated across all severity categories. Interferon (IFN)-α, IFN-γ, IL-2, IL-7, IP-10, IL-12p40, MIG, eotaxin, and tumor necrosis factor (TNF)-α concentrations were not significantly elevated for any severity category.

Discussion

From serological examination of cytokine responses to human MPXV infection, a cytokine storm appears to occur during human monkeypox disease. We also found evidence of a prominent T helper 2 (Th2) response, and a dampened Th1 response, following MPXV infection. Th2-associated cytokines IL-4 (and the closely related IL-13), IL-5, and IL-6 were elevated above normal human range and IL-10 was elevated in serious cases, whereas serum concentrations of Th1-associated cytokines (IL-2, TNF-α, IL-12, IFN-γ) fell within normal range for all severity categories. During MPXV infection, Th2 immune responses (e.g., IL-10, IL-4) could downregulate Th1-immune responses (e.g., IL-12, INF-γ, IL-2) as has been seen during infection with recombinant vaccinia virus expressing IL-4 [8]. IL-4, IL-10, and IL-13 increase vaccinia virus replication in patients with atopic dermatitis and in mice deficient in Th1 cytokines challenged with vaccinia virus [8], [9], [10]. IL-10 release is often a strong indication of cytokine storm. IL-10 dampens the inflammatory response by downregulating the function of neutrophils, monocytes, and dendritic cells. In addition, IL-10 reduces the production of Th1 cytokines, and promotes the development of regulatory T cells (Treg) and the survival of B cells [11], [12], [13], [14], [15]. Although we did not directly assay for a Treg response, the data suggest that further work to elucidate types of T cell responses elicited by MPXV in humans is necessary as these responses may be key contributors to pathogenesis. Alternatively, orthopoxvirus inhibitors of TNF-α, IFN-α, and IFN-γ have been described previously, and our data suggest that immunomodulation of these cytokines might occur during human MPXV infections.

We determined that a certain subset of cytokines were associated with disease severity. MIP-1α and MIP-1β were significantly elevated in cases of mild disease compared to moderate and severe cases. MIPs are pro-inflammatory chemokines which are produced during the innate immune response and promote the migration of granulocytes to areas of infection. GM-CSF, IL-10, and sIL-2R were present in extremely high concentrations in serum samples from serious disease cases. Elevated IL-10, GM-CSF and sIL-2R serum concentrations, in association with tempered IL-2, IFN-γ, and IL-12 responses are commonly indicative of a Treg response and a dampening of the immune response [13], [16], [17], [18], [19], [20], [21], [22], [23]. Viruses such as HIV and HCV modulate the host immune system to promote the expansion of Tregs, thereby facilitating virus persistence through immune subversion [24]. These data suggest that MPXV may promote similar immune-subversion mechanisms to enable persistence and continued spread, which may result in more serious clinical disease.

Study limitations include time point after exposure that the serum samples were obtained and influence of other microbial agents on immune response to MPXV infection. As serum samples were taken from symptomatic patients (fever, rash) during a surveillance program, duration of infection is unknown but can be assumed to be at least 4 days post-exposure. Also, we cannot rule out the presence of other bacterial, parasitic, or viral agents endemic to the DRC. Despite these limitations, we were able to use cytokine-profiling techniques to assess immunological correlates of monkeypox disease in humans. Our data revealed potential indicators or factors for development of serious disease. Future studies should build on this work to assess these potential biomarkers for serious disease and/or targets for intervention. These data will also be useful in the evaluation of animal models. A major shortcoming for animal models is the lack of human data for comparison. Our study is the first step in compiling human data on immunological correlates of monkeypox disease that can evaluate the relevance of animal models to the human condition.

Funding

This work was supported by the Defense Threat Reduction Agency [Project # 195726]. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army.

Competing interests

None declared.

Ethical approval

Ethical approval for the original surveillance study was obtained from the participating institutions, and informed consent was obtained from all participants.