The immunosuppressive activity of artemisinin‐type drugs towards inflammatory and autoimmune diseases

The sesquiterpene lactone artemisinin from Artemisia annua L. is well established for malaria therapy, but its bioactivity spectrum is much broader. In this review, we give a comprehensive and timely overview of the literature regarding the immunosuppressive activity of artemisinin‐type compounds toward inflammatory and autoimmune diseases. Numerous receptor‐coupled signaling pathways are inhibited by artemisinins, including the receptors for interleukin‐1 (IL‐1), tumor necrosis factor‐α (TNF‐α), β3‐integrin, or RANKL, toll‐like receptors and growth factor receptors. Among the receptor‐coupled signal transducers are extracellular signal‐regulated protein kinase (ERK), c‐Jun N‐terminal kinase (JNK), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K), AKT serine/threonine kinase (AKT), mitogen‐activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) kinase (MEK), phospholipase C γ1 (PLCγ), and others. All these receptors and signal transduction molecules are known to contribute to the inhibition of the transcription factor nuclear factor κ B (NF‐κB). Artemisinins may inhibit NF‐κB by silencing these upstream pathways and/or by direct binding to NF‐κB. Numerous NF‐κB‐regulated downstream genes are downregulated by artemisinin and its derivatives, for example, cytokines, chemokines, and immune receptors, which regulate immune cell differentiation, apoptosis genes, proliferation‐regulating genes, signal transducers, and genes involved in antioxidant stress response. In addition to the prominent role of NF‐κB, other transcription factors are also inhibited by artemisinins (mammalian target of rapamycin [mTOR], activating protein 1 [AP1]/FBJ murine osteosarcoma viral oncogene homologue [FOS]/JUN oncogenic transcription factor [JUN]), hypoxia‐induced factor 1α (HIF‐1α), nuclear factor of activated T cells c1 (NF‐ATC1), Signal transducers and activators of transcription (STAT), NF E2‐related factor‐2 (NRF‐2), retinoic‐acid‐receptor‐related orphan nuclear receptor γ (ROR‐γt), and forkhead box P‐3 (FOXP‐3). Many in vivo experiments in disease‐relevant animal models demonstrate therapeutic efficacy of artemisinin‐type drugs against rheumatic diseases (rheumatoid arthritis, osteoarthritis, lupus erythematosus, arthrosis, and gout), lung diseases (asthma, acute lung injury, and pulmonary fibrosis), neurological diseases (autoimmune encephalitis, Alzheimer's disease, and myasthenia gravis), skin diseases (dermatitis, rosacea, and psoriasis), inflammatory bowel disease, and other inflammatory and autoimmune diseases. Randomized clinical trials should be conducted in the future to translate the plethora of preclinical results into clinical practice.

The criteria to extract the relevant literature for this focused review article were as follows: The PubMed database was screened for articles related to artemisinin/artesunate and inflammatory/autoimmune diseases until July 30, 2020. Some exclusion criteria were defined as follows: Combination therapies of artemisinin and its derivatives and other established or investigational drugs were excluded. Only experimental studies regarding monotherapies with artemisinin-type drugs were considered in the present review article. Another exclusion criterion was the focus on the anti-inflammatory activity independent of infectious diseases. As infectious diseases caused by microbial pathogens may be accompanied by inflammation. In such cases, the anti-inflammatory activity of artemisinins cannot be clearly separated from their anti-infective effects. Therefore, we exclusively focused on anti-inflammatory and autoimmune disease without contribution of communicable diseases to solely and undoubtedly elaborate the immunosuppressive molecular mechanisms.
Looking into the future, another intention of this review was to provide comprehensive preclinical information stimulate the conductance of clinical trials to prove the therapeutic potential of artemisinin and its derivatives in clinical settings.

| RHEUMATIC DISEASES
Among the long list of rheumatic disorders, artemisinin derivatives (artemisinin, artesunate, artemether, dihydroartemisinin, and the semisynthetic derivatives DC32, SM 903, and SM934) have been investigated in rheumatic arthritis, osteoarthritis, and osteoporosis as well as lupus erythematosus and gout (Table 1).

| Rheumatoid arthritis
Rheumatoid arthritis is a frequent inflammatory disease of the joints associated with pain, swelling, and stiffness of the joints. 39 This is an autoimmune disease, which affects Caucasians less than other populations. Mis-regulated immune cells migrate into the joints and release proinflammatory cytokines (e.g., interleukin-1β [IL-1β], IL-6, tumor necrosis factor-α [TNF-α]) leading to osteoclast activation and cartilage inflammation. The function of regulatory T cells (Treg) is dampened in dependency of forkhead box P-3 (FOXP-3) activity and elevated TNF-α levels.
Synovitis is also frequently observed. Cytokines also induce tumor-like proliferation at the synovia. This tumor-like tissue is called pannus, which contributes to the destruction of bone and cartilage by necrosis-inducing collagenases and other proteolytic enzymes. Rheumatoid arthritis-specific fibroblasts spread from the synovia into the body via the blood vessel system and lead to a generalization of the disease.
Therapy options are nonsteroidal antiphlogistic drugs (NSAIDs) and glucocorticoids against inflammation, disease-modifying anti-rheumatic dugs (DMARDs), including methotrexate (as gold standard), azathioprine, hydroxychloroquine, and the therapeutic biologicals anakinra, tocilizumab, adalimumab, infliximab etc.) as well as analgetic drugs against pain. Rheumatoid arthritis is difficult to be treated, because many patients do not respond and high side effects hamper treatment success (e.g., diarrhea, nausea and vomiting, gastrointestinal bleeding, headache, dizziness, edema, increased risk for ulcers). Therefore, novel therapeutic strategies are urgently required and artemisinin-type drugs may offer promising alternatives. 40,41 The majority of experimental data have been raised in animal models of rheumatoid arthritis with wellestablished models such as type II collagen-induced arthritis (CIA) or Freud's complete adjuvant (FCA)-induced arthritis in mice or rats (Table 1). [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] Some authors also applied experimental in vitro settings by using lipopolysaccharide (LPS)-stimulated murine macrophages or fibroblast-like synoviocytes isolated from rheumatoid arthritis patients. 16,21 Synovial tissue biopsies from rheumatoid patients also have been investigated. 7 Using appropriate experimental models, which are established in the scientific community of rheumatoid research, represents an important prerequisite to estimate the therapeutic effect of artemisinin-type drugs in an objective manner.
T A B L E 1 Anti-inflammatory activity of artemisinin-type drugs against rheumatic diseases At the phenotypic level in vivo, which compares to some extend to the clinical symptoms of patients, artemisinin-type drugs led to improvements. We did not find papers reporting no effect or worsening of rheumatoid arthritis symptoms. Symptomatic improvements of the disease were decreased joint and food pad inflammation and swelling, decreased pannus formation, reduced cartilage and bone erosion, reduced paw edema as well as improved functional parameters (stair climbing ability, motility, suppression of mechanical allodynia) and improved clarity of articular cavity (Table 1). [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] At the microscopic level, reduced proliferation of fibrous connective tissue, chondrocytes, and blood capillaries were observed upon treatment with artemisinins. At the molecular level, a plethora of results provided details of the molecular mechanisms and signaling pathways involved in the anti-rheumatic activity of artemisinin and its derivatives (Table 1). These compounds inhibited the secretion of proinflammatory cytokines and chemokines, that is, TNF-α, IL-1β, IL-6, IL-8, IL-17a, C-X-C chemokine ligand (CXCL-12), CX3CL-1, the leucocyte activator RANTES and the macrophage activator monocyte chemoattractant protein 1 (MCP-1). (COX-2) is a well-known mediator of inflammation, which is induced upon exposure of cells to TNF-α and IL-1β. COX-2 is upregulated in rheumatoid arthritis, and artemisinin-type drugs downregulated its activity. Several signal transduction pathways involved in inflammation were downregulated by these drugs, for example, the expression and/or phosphorylation of signal transducers (e.g., AKT serine/threonine kinase (AKT), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), extracellular signal-regulated protein kinase [ERK], and activation and/or nuclear translocation of transcription factors (e.g., retinoic-acid-receptor-related orphan nuclear receptor γ [ROR-γt], nuclear factor κ B [NF-κB] as important inflammatory regulator and FOXP-3, which is important for T-cell regulation). Oxidative stress represents an important trigger of inflammation. Artemisinin and its derivatives counteracted oxidative stress in rheumatoid arthritis by upregulating NF E2-related factor-2 (NRF-2), kelch like ECH associated protein 1 (KEAP-1), and heme oxygenase 1 (HO-1) and downregulating HIF-1 α). Matrix metalloproteinases are expressed in inflammatory conditions and regulate the bioavailability and activity of inflammatory cytokines. Matrix metalloproteases (MMP-2) and MMP-9 were downregulated upon exposure to artemisinin-type drugs. Furthermore, artemisinin and its derivatives induced apoptosis and autophagy.
In summary, the synopsis of published data reveals quite an interesting picture that artemisinin and its derivatives were indeed able to inhibit inflammation in experimental settings of rheumatoid arthritis.

| Lupus erythematosis
Lupus erythematosus is an autoimmune disease characterized by a hyperactive immune system, which attacks healthy tissues. The disease typically affects joints skin, kidney, blood cells, heart, and lungs. The most common and severe form is systemic lupus erythematosus (SLE). 42,43 SLE therapy comprises of methotrexate, immunosuppressive drugs (corticosteroids, hydroxychloroquine, and antibody therapy (e.g., belimumab, which inhibits the B-cell activating factor, BAFF). 44,45 Clinical symptoms of lupus erythematosus improved after application of artemisinin-type drugs, that is, the survival of animals increased, lymphadenopathy was reduced, the glomerulonephritic lesions, the 24 h urinary proteins levels, the renal and serum anti-dsDNA antibodies, and the serum creatinine levels all decreased (Table 1). [24][25][26][27][28][29][30][31][32][33][34][35] Considerable immune cell alterations were observed, for example, the Tfh cell counts in the spleen and the effector/memory T-cell counts decreased, while the Treg and quiescent B cell numbers and the plasma cell differentiation increased. The LPS-induced spleen cell proliferation also went down ( Table 1).
The decrease in cellular senescence and senescence-associated secretory phenotype was linked to reduced expression of p53, p21, and p16. Th1 cell-related anti-dsDNA IgG2a and IgG3 antibody production was suppressed, while Th2-cell-related anti-dsDNA IgG1 generation was activated. Proinflammatory cytokine production was  (Table 1).
In sum, these data show that artemisinin and derivates corrected the immune cells and cytokine imbalances and related deregulations in signal transduction leading to SLE.

| Arthrosis and gout
While rheumatic arthritis and lupus erythematosus are inflammatory diseases without tissue destruction, osteoarthritis and arthrosis belong to the destructive rheumatic diseases. Gout is categorized as metabolic disease associated with rheumatic complaints. 46 A main factor of arthrosis is the deposition of urate crystals in the joints. Arthrosis is associated with osteoporosis and painful joint deformation. Drug therapy comprises of glucocorticoids, nonsteroidal antiphlogistic drugs, and others. The therapeutic effects of artesunate include a decrease of collagen content and reduced fibroblast viability and proliferation and increased apoptosis at the same time. Associated ERK and PI3K/AKT/mTOR and AMPK/mTOR signaling was also suppressed by artesunate (Table 1). 36,37 Inflammatory arthritis with swollen joints is also indicative for gout. This disease is caused by elevated uric acid levels. The uric acid crystals deposit in joints, tendons, and surrounding tissues and cause the typical gout attacks.
These crystals provoke inflammatory reactions of macrophages, which are initiated by the nucleotide-binding domains and leucine-rich repeat pyrin domains containing 3 (NLRP-3) inflammasome protein complex. NLRP-3 binds and activates caspase 1, which cleaves pro-IL-1β to IL-1β as main player in the inflammatory cascade. Gout treatment consists of application of NSAIDS, glucocorticoids, low-dose colchicine, as well as allopurinol, probenecid and the IL-1 inhibitor canakinumab, all of which are associated with side effects. 47 Artemisinin-type drugs have been not well analyzed in gout as of yet. In monosodium urate-induced gout in mice, food and ankle swelling was reduced by artemisinin and NLRP-3 inflammasome activation was suppressed.
This was associated with a decrease in IL-1β and caspase 1 ( Table 1). 38 As numerous encouraging results are available for the other rheumatic diseases discussed above, more investigations on the therapeutic potential of artemisinin in gout should be encouraged.

| Asthma
Asthma represents a long-term airway inflammation with airflow obstruction and bronchospasms. Affected patients suffer from symptoms such as coughing, wheezing, shortness of breath, and chest tightness. Chronically inflamed bronchi and bronchioles induce increased contractibility of the surrounding smooth muscles, which narrows the airways. Participating immune cells in this process are eosinophils, T lymphocytes, macrophages, and neutrophils.
Asthma is reversible (in contrast to chronic obstructive pulmonary disease, COPD). 48 Asthma cannot be cured.
In conclusion, there are numerous in vitro and in vivo results demonstrating that artmisinin-type drugs have the potential to treat asthma.

| Acute lung injury and pulmonary fibrosis
Acute lung injury or pneumonia represents a systemic inflammatory response in the lungs with short breath and bluish skin coloration without participation of heart failure. Lungs may collapse and low oxygen blood levels may occur. Immune cells (neutrophils, T-lymphocytes, and eosinophils) migrate into the inflamed lung tissue and enhance the phenomenon. 70 The therapeutic value of corticosteroids and NO is not established and critically discussed. 71 The treatment with artemisinin and its derivatives of acute lung injury in experimental animal models de-  Table 2). [62][63][64][65][66][67][68] Among the symptoms of pulmonary fibrosis are short breathing, dry cough, respiratory failure, pneumothorax, and others. 72,73 A typical feature is that normal lung tissue is transiently and irreversibly exchanged with fibrotic scarred tissue. Curative therapies are not available and current treatment concepts aim to slow down the progression of scar development by pirfenidone to downregulate growth factors and procollagens 1 and 2 and by corticosteroids as immunosuppressive therapy. 74 A well-known side effect of cancer therapy with bleomycin is the occurrence of lung fibrosis. Therefore, bleomycin can be experimentally used to mimic pulmonary fibrosis in vivo. Treatment of bleomycin-induced lung fibrosis with dihydroartemisinin was characterized by decreased levels of TGF-β1, TNF-α, α-smooth muscle actin (α-SMA), and NF-κB in lung tissues. Reductions of collagen synthesis and fibroblast proliferation also improved the symptoms pulmonary fibrosis ( Table 2). 69 4 | NEUROLOGICAL DISEASES

| General neuroinflammation
Among other factor, neuroinflammation may be caused by autoimmune reactions or mechanic brain traumata.

EFFERTH AND OESCH
| 3037 ERK and MAPK pathways. A number of proinflammatory mediators were also suppressed (NO, iNOS, MyD-88, PGE-2, COX-2, and mPGES) ( Table 3). [76][77][78][79][80][81]92 4.2 | Autoimmune encephalitis, alzheimer's disease, and myasthenia gravis Autoimmune encephalitis is a heterogeneous group of neurological disorders associated with changes in consciousness, cognitive decline, seizure, abnormal movements. 93,94 Experimental models to test the activity of artemisinin-type drugs included treatment with MOG35-55 and specific genetically modified mouse strains.  (Table 3). [82][83][84][85][86] The appearance of neurofibrillary tau protein complexes and β-amyloid plaques is typical in Alzheimer's disease. 95 Furthermore, neuroinflammation is also involved in this disease. 96 Microglia cells activated by proinflammatory cytokines are unable to clear up β-amyloid, which leads to plaque formation. Increased IL-1β production is associated with decreased synaptopysin contents and synaptic loss. Satisfying drug treatments are not yet available. 97 There are also clues that artemisinin and its derivatives may be helpful for the treatment of Alzheimer's disease.
These compounds improved spatial memory, the neuritic plaque burden decreased pathological features in hippocampus and cortex improved and the β-secretase activity went down upon treatment. Furthermore, NALP-3 inflammasome activity was inhibited, and the amyloid polypeptide (hIAPP) was disaggregated and inhibited.
The disease results from antibodies that lead to the decoy of nicotinic acetylcholine receptors between muscles and nerves, thus preventing muscle contraction. 98 Drugs to treat myasthenia gravis are acetylcholine esterase inhibitors (neostigmine and pyridostigmine) and immunosuppressants (prednisone and azathioprine). 99 Investigations on artemisinins to treat myasthenia gravis are still in its infancy. Nevertheless, some hints are available. Artemisinin and artesunate decreased lymphocyte proliferation, modulated Th1/Th2 cytokine expression, increased Treg cell counts, and the production of anti-R97 immunoglobulin antibodies (Table 3). 90 Typical symptoms were reduced or disappeared by artemisinins in dinitrochlorobenzene (DNCB)-, tetradecanoylphorbol-acetate (TPA)-or concavalin A-induced dermatitis in animals (e.g., decreased dermatitis scores, ear, spleen, and lymph node weights, skin epidermal thickness as well as decreased IgE levels). The tissue infiltration of inflammatory cells was reduced, the counts of Th7 cells decreased, while that of Treg cells increased.

| Rosacea and psoriasis
Rosacea is an autoimmune disease which affects Caucasians more than other populations. Typical symptoms are pimples, skin swelling, redness, and others. 111  The available therapeutic options may relieve the symptoms of psoriasis, but do not lead to satisfying curative effects. Treatment is attempted with steroids and NSAIDs, methotrexate, UV-light plus psoralen, the TNF-αinhibitory antibodies infliximab and adalimumab and the TNF-α decoy receptor eternacept. Efalizumab and alefacept are also used, which are directed against T-cells. 114 Therapeutic options for psoriasis have not been investigated in detail yet. Imiquimod-induced experimental psoriasis was treated with artesunate leading to beneficial results: systemic inflammation, T-cell numbers in draining lymph nodes, cumulative score, epidermal thickening, and proliferation rate (Ki-67 expression) all went down upon artesunate treatment (Table 4). 110

| INFLAMMATORY BOWEL DISEASE
Ulcerative colitis and Crohn's disease are common forms of inflammatory bowel disease (IBD), which occur in the colon and small intestine. Typical IBD symptoms are ulcers, abdominal pain, diarrhea, rectal bleeding, weight loss, and others. Multiple pathophysiological factors lead to inflammatory responses. 115,116 Several treatment strategies have been recommended for IBD, for example, mesalazine, immunosuppressants (prednisone azathioprine, methotrexate, and TNF-α inhibitors), which partly can exert harsh adverse effects. 117 There are many results showing improvements of clinical IBD symptoms by treatment with artemisinin-based drugs, for example, the body weight regained towards normal levels, the disease activity index was improved, colonic shortening was stopped, the gastric juice parameters normalized, and diarrhea and rectal bleeding were T A B L E 5 Anti-inflammatory activity of artemisinin-type drugs against inflammatory bowel disease   (Table 5). [118][119][120][121][122][123][124][125][126][127][128] At the mechanistic level, the release of several proinflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-12, IL-17) was suppressed by artemisinin and its derivatives, while the release of IL-10 as anti-inflammatory cytokine was boosted. The activity of COX-2 as inflammatory mediator was suppressed. IBD-related apoptosis in colonic tissues was reduced as indicated by reduced expression of BAX, caspase 9 and phosphorylated p38 MAPK.
Inflammation-related oxidative stress was also reduced by artemisinin and its derivatives, because the expression and activity of SOD, MDA MPO and glutathione decreased, while that of HO-1 increased. IBD-associated signaling was considerably affected by artemisinin and its derivatives. The signaling routes of NF-κB, TLR-4, RANK/RANKL, PI3K/AKT, FRA, and NF-ATC1 were all downregulated (Table 5).

| OTHER DISEASES
Kidney inflammation (nephritis) can affect tubules, glomeruli (glomerulonephritis), and the interstitial tissue inbetween (interstitial nephritis). [129][130][131] SLE can be associated with lupus nephritis. The inflammatory processes cause reduced renal blood flow and thus reduced urine production. Hematuria and proteinuria may also result from renal inflammation.
In animal models, an improvement of renal function associated with a decrease of tubulointerstitial inflammation, fibrosis, and proteinurea was reported. A number of proinflammatory cytokines and chemokines Autoimmune hepatitis  (Table 6). [132][133][134][135]145 Type 1 diabetes mellitus is characterized by a lack of pancreatic insulin production in juvenile patients.
Liver inflammation can be acute or take a chronic course. A specific chronic form is autoimmune hepatitis, which is an autoimmune reaction directed against hepatocytes. Common auto-antibodies are the anti-nuclear antibody (ANA), smooth muscle antibody (SMA), and atypical perinuclear antineutrophil cytoplasmic antibody (p-ANCA).
Patients with autoimmune hepatitis have an increased risk for the development of liver cirrhosis and liver cancer. 147 Artesunate has been used to treat concavalin A-induced autoimmune liver injury. 140,141 As a result, the liver transaminases in the serum decreased, less proinflammatory cytokines (TNF-α, IFN-γ, IL-6, IL-17) and more antiinflammatory cytokines (IL-10) were determined, and the phosphorylation of several signal transducers (ERK, JNK, p38 MAPK, NF-κB p65, and IκB-α) was inhibited (Table 6).
In autoimmune thyroiditis, antibodies against thyroid cells are produced, which destroy these cells. 148 Dihydroartemisinin treatment inhibited antithyroglobulin antibody and thyroid peroxidase antibody production in a mouse model of autoimmune thyroiditis. 142 This observation was associated with a normalization of the Th1/Th2 imbalance, decreased calcium flow by CXCL-10 binding to CXR-3 and a suppression of expression and phosphorylation several signaling molecules (PI3K, AKT, and NF-κB) ( Table 6).
Multiorgan failure by hemorrhagic shock is also associated with inflammatory processes. 151,152 Although the experimental evidence for the activity of artemisinin-type compounds is still weak in this context, artesunate improved multiple organ injury and dysfunction in an experimental rat model of hemorrhagic shock. 144 This effect was correlated with a decrease of the proinflammatory TNF-α and IL-6, decreased activities of endothelial and inducible nitric oxide synthases (eNOS, iNOS) as well as of NF-κB (Table 6).

| TOXICOLOGY OF ARTEMISININ-TYPE DRUGS
Although the toxicity of artemisinins have been well-investigated in preclinical and clinical malaria studies and this type of drugs are generally accepted to be relatively safe, toxicological issues are still in the focus of interest. Since artemisinins have been recently considered for other diseases than malaria, this issue has to be considered again. This is true for inflammatory and autoimmune diseases as discussed in the present review article but also for all other discussed disease indications (e.g., cancer schistosomiasis, trypanosomiasis etc.), where longer treatment durations might be necessary to observe clinically visible therapeutic effects. Therefore, we give here an update of the past decade on our comprising review on the toxicity of artemisinin-type drugs. 153

| Neurotoxicity
Among the most reported toxicities regarding therapeutic use of artemisinins is neurotoxicity, which is dependent of the duration of treatment. In animal experiments, behavioral changes, balance and coordination disturbances, eye reflex and auditory loss were investigated. The lowest observed neurotoxic effect levels (LONEL) of artheether have been determined in the plasma of monkeys, rats, and dogs. 154 The authors observed that the exposure time to provoke minimal neurotoxicity was longest in monkeys and shortest in rats, indicating that LONEL values are lower in primates (including human beings) than in rodents. Another observation was that orally administered artelinic acid needed longer exposure times to provoke neurotoxicity than intramuscularly applied arteether, which indicates that the application route and the water solubility of artemisinin-type drugs play an important role. In dogs, artemether EFFERTH AND OESCH | 3045 induced neuropathic CNS changes with motor and motor neuron damages, but no disturbances in behavior.
By contrast, artesunate did not provoke neurotoxicity. 155 Neurotoxicity was rarely seen in malaria patients, which is probably due to the fact that the common duration of therapy for only 3-5 days seems to be safe.

| Hematotoxicity
Hematotoxicity can appear in the erythropoietic and leukopoietic differentiation lineages, and erythropoietic toxicities by artemisinins are much better documented that the leukopoietic one. 153 If disturbances occur in erythropoiesis in maternal or fetal organisms, embryotoxicity may appear (see below). A frequently used parameter for erythrocytic toxicity is the determination of reticulocyte count. Reticulocytes in the peripheral blood are bone marrow-derived precursor cells. Reduced reticulocyte counts frequently occur, but mostly with mild to moderate courses, which are reversible after the end of therapy. Leukopoietic lineage: Artesunate induced clastogenic and aneugenic effects caused by oxidative and nitrosative stress and thereby caused apoptosis in human lymphocytes. 160 While artemisinins alone or malaria-related ACTs may cause only mild to moderate hematotoxicity, this cannot be taken for granted for all combination therapies.
The compassionate application of dichloroacetate and artesunate by alternative practitioner resulted in the death of a glioblastoma patient. The patient experienced bone marrow toxicity (leukopenia and thrombocytopenia) and hepatotoxicity and died 10 days after the infusion. 161

| Embryotoxicity and teratogenicity
The inhibition of erythropoiesis fired discussions on a potential risk for embryotoxicity of artemisinin-type drugs. 162 However, embryos may get lost during the first weeks of pregnancy without becoming aware to the women knowing about their pregnancy in this early stage. Malformation of damaged but surviving animals can also not be determined with convincing certainty. Therefore, WHO warned on the use of artemisinins within the first three trimester. 163 The potential embryotoxic risk is further aggravated by the fact that embryotoxic effects of artemisinins have been documented in preclinical studies in mice, rats, rabbits, and monkeys. 153 A recent investigation confirmed the embryotoxic risk of artesunate at high doses (8 mg/kg) in rats regarding several parameters: the gravid uterine weight and fetus weights decreased, the number of fetal deaths and postimplantation losses increased. There were also more malformations of the embryos. 164 Therefore, embryotoxicity is an important aspect to be considered if it comes to the development of novel artemisinin derivatives. The synthetic tetraoxane drug candidate (RKA182) and a trioxolane equivalent (FBEG100) revealed embryotoxicity and erythroblasts depletion in rodents. 165 The novel derivative artefenomel and artesunate as positive control drug were similarly embryotoxic in rats regarding cardiovascular defects and fetal resorption. Both compounds depleted embryonic erythroblasts and reduced maternal reticulocyte counts. However, artefenomel was ∼250-fold less embryotoxic in vitro than dihydroartemisinin, the active metabolite of artesunate. 166 Lipid nanoparticles containing artemether and clindamycin caused ∼90% fetal resorptions, but no postimplantation losses or fetal malformations.
Nevertheless, should this nanoparticle formulation be further developed as drug, its use in pregnancy must be excluded. 167

| Cardiotoxicity
Cardiotoxicity is a well-known side effects of anthracyclines because of the generation of radical oxygen species (ROS), which cannot be sufficiently detoxified in cardiac tissues. As ROS are also produced by artemisinin-type drugs, the question arises whether or not cardiotoxicity also applies for this drug class. Cardiotoxicity has been reported in animals, while clinical trials in human malaria patients did not show alerting signs of cardiotoxicity in the past.
In dogs, artemether provoked a prolongation of frequence-correct Q-wave T-wave interval (QTc) intervals on electrocardiograms, while artesunate decreased the heart rate. 155 In another study, artesunate decreased the heart rate in vitro, but not in vivo. 156 In a Zebrafish model to study cardiotoxicity, low dose artesunate protected from verapamil-induced heart failure. 168 Cardiac malformation was prevented by artesunate. Furthermore, Verapamil-induced venous stasis, cardiac output decrease, and blood flow dynamics reduction were restored by artesunate in zebrafish. Monitoring of biomarkers for cardiotoxicity by RNA-sequencing revealed that the gene for frizzled receptor 7a (fzd7a) was upregulated in zebrafish with verapamil-induced heart failure but downregulated upon artesunate. As frizzled receptors belong to the Wnt signaling pathway, artesunate may confer cardioprotection via this pathway. By contrast, high artesunate concentrations exerts cardiotoxic effects. While this kind of biphasic effects are well documented in the literature and referred to as hormesis, 169,170 this is the first report for hormetic effects of artemisinin-type drugs.

| Other toxicities
Nephrotoxicity: While renal failure and tubular necrosis have been occasionally reported with artemisinins in the past, 171 another previous report on histopathological examinations of adult rodents treated with artemether-loaded lipid nanoparticles did not find signs of nephrotoxic changes. 172 Gastrointestinal toxicity: Upon artesunate application, the gastric pH increased, and the volume of gastric secretions decreased at supra-therapeutical doses. 173  clobazam. The patient suffered from a dramatic increase of liver enzymes. This reaction was reversible upon cessation of artesunate and the Chinese herbal decoction. 176 Reproductive toxicity: In healthy rats not suffering from malaria, artemisinin suppressed the levels of folliclestimulating hormone, while it increased those of progesterone. 177 In addition to hormonal imbalance in female organisms, artesunate also exerted reproductive toxicity in males. Artesunate decreased epididymal sperm count and increased the number of sperms with abnormal head morphology. In addition, spermatozoa of artesunatetreated mice had significantly more DNA strand breaks than untreated control animals, indicating that artesunate crossed the blood-testis barrier. 178 Reduction of toxicity induced by xenobiotic compounds: Interestingly, artemisinin-type drugs have not only been subject to unravel toxic reactions but also whether or not this drug class is capable to reduce the toxicity induced by other xenobiotic compounds such as anticancer drugs. This is a new aspect in toxicological research on artemisinins, which deserves further attention in the future. Doxorubicin caused hyperemia and hemorrhages in the liver and heart of rats, which was associated with increased caspase-3, TNF-α, iNOS, and NF-κB expression. Artemisinin suppressed the expression of these proteins, indicating cardio-and hepatoprotective effects of artemisinin. 179 Activated NRF2 signaling is related with tissue-protection. Artemisitene is an NRF-2 inducer, which protected from bleomycin-induced lung injury. 180

| SYNOPSIS OF DATA AND FUTURE PERSPECTIVES
There is no doubt that the spectrum of bioactivities of artemisinin-type drugs is much broader than initially expected. This type of compounds is not only active against malaria but also other infectious and vector-borne diseases as well as cancer. [181][182][183][184] In the present review, the focus is on inflammatory and autoimmune diseases.
The question may arise, whether or not such broad spectrum of activities is realistic, and if so, how specific such drugs could be in terms of disease relevant target proteins as underlying molecular mechanisms. There is an overwhelming number of publications pointing to the transcription factor NF-κB as possible common mode of action of artemisinin and its derivatives, because NF-κB plays a role in many of the diseases, where artemisinins exert inhibitory activity. As a matter of fact, NF-κB is involved in the pathology of viral diseases, 5,[184][185][186][187] cancer, 188,189 and even malaria. [190][191][192] The central role of NF-κB for innate and acquired immune functions is wellstudied. It regulates the expression of proinflammatory cyto-and chemokines and thereby drives the activation, proliferation, and differentiation of immune cells. 193,194 A majority of published literature indeed speaks for the inhibition of NF-κB as major mechanism explaining the activity of artemisinin and its derivatives against inflammatory and autoimmune diseases (Figure 1). NF-κB is a dimer formed by several protein that share a Rel binding domain. Known proteins with a Rel domain are NF-κB1 (p50 and p105), NF-κB2 (p52 and p100), RelA (p65), and RelB: The NF-κB signaling is a tightly controlled process. In the cytoplasm, NF-κB is inactive and complexed with IκB. IκB kinase (IKK) phosphorylates IκB-α, which is thereby degraded. Cleavage of the NF-κB/IκB complex activates NF-κB, which is then translocated to the nucleus. Numerous regulatory gene sequences contain a κB motif. Binding of the NF-κB dimer to this binding motif actives the transcription of NF-κB downstream genes.
In rare cases, repression of transcription has been observed. Many of the NF-κB regulated genes are involved in regulating the immune system. 195 A synopsis of all published studies on the activity of artemisinin-type drugs against inflammatory and autoimmune diseases allows to compile a hypothetical network, how artemisinin and its derivatives inhibit signaling in inflammatory and autoimmune diseases ( Figure 2). There are numerous receptor-coupled signaling pathways that have been described to be inhibited by artemisinins, including the receptors for IL-1, TNF-α, β3-integrin, or RANKL as well as toll-like receptors and growth factor receptors. Among the signal transducers are well-known molecules such as ERK, JNK, PI3K, AKT, MEK, phospholipase C γ1 (PLC-γ1), and others. All these receptors and signal transduction molecules are known to contribute to the inhibition of NF-κB. This network compilation also demonstrates that artemisinin-type drugs may not only inhibit NF-κB by direct binding but by silencing the up- proliferation-regulating genes, signal transducers, and genes involved in antioxidant stress response. It is important to mention that NF-κB is not the only transcription factor whose activity is inhibited by artemisinin and its derivatives. These drugs also inhibit the activity of mTOR, activating protein 1 As outlined in the Section 1, we explicitly excluded inflammatory conditions related to infectious diseases from the literature survey. The intention was to separate anti-inflammatory of artemisinins from their antimicrobial activity, because microbial infections frequently also cause inflammation. The cure from microbes and other parasites by artemisinins may lead to a reduction of accompanied inflammation in affected tissues. However, from a practical point of view it does not matter that much, whether or not anti-inflammatory treatment effects of artemisinin-type drugs occur in a primary or secondary manner. In fact, there are several reports reporting on the improvement of inflammation in the course of infectious diseases. After having dissected the anti-inflammatory molecular mechanisms of artemisinins in the chapters above, it is now interesting to have a look at infectious conditions that simultaneously cause inflammations. Some authors reported on the improvement of nephritis in the course of malaria treatment. [196][197][198] A myasthenia gravis patient suffering from severe malaria experienced favorable outcome for both conditions by artesunate treatment. 199 This anti-inflammatory effect has not only been observed for Plasmodia but also for other protozoa such as Naegleria fowleri, which causes meningoencephalitis. 200,201 Comparable anti-inflammatory effects of artemisinins also have been described for bacteria-induced arthritis 202  Another aspect to be considered is that artemisinin and its derivatives would probably not used as monotherapy for the treatment of inflammatory and autoimmune diseases, if they ever reach clinical routine. Rather, they would be combined with other established drugs. Therefore, it is interesting to know whether or not drug combinations involving artemisinins act in an additive or synergistic manner, or whether even antagonistic effects have to be considered. There are only very few hints as yet for additive or synergistic immunosuppressive effects.
The combination of artesunate with an immunomodulator (methotrexate/triptolide/azathioprine) exhibited superior induction of macrophage apoptosis than each drug applied alone. 205 There are a few more clues from the literature that artemisinin-based combination treatments (ACT) may result in synergies to treat malaria, for example, the approved artemether-lumefantrine combination. 206 An ACT against malaria containing methylene blue as investigational drug as well as the combination of artesunate and curcumin were also described in this context. 207,208 The combination of artesunate with hydroxychloroquine improved renal function in immunoglobulin A-induced nephropathy. 209,210 Synergies of artemisinin-type drugs are also known for chemotherapy, radiotherapy, antibody therapy of cancer cells, [211][212][213][214][215][216] and anti-schistosomiasis therapy. 217 Drug combinations may not always be beneficial, and in rare cases toxic reactions may be provoked. 218 These few examples of potential synergistic or even antagonistic reactions of ACTs highlight the importance also to investigate drug combinations with artemisinins for inflammatory and autoimmune diseases in the future.
During the past two decades, many groups including our own scientifically substantiated the wisdom of TCM handed over for millennia by (1) identification of the bioactive chemical compounds in medicinal plants (e.g., artemisinin in Artemisia annua) and (2) unraveling the molecular and cellular mechanisms of Chinese herbal medicine with modern scientific methods. [219][220][221][222][223][224] Based on the achievements in phytochemistry and phytopharmacology, the isolated chemical structures may also serve as lead compounds the development of novel (semi)synthetic derivatives. A plethora of artemisinin derivatives have been already described in the literature. It is reasonable to assume that specific artemisinin derivatives are not only active against diverse inflammatory and autoimmune diseases, but that different derivatives may be identified with specific activity against each different disease.
Novel artemisinin derivatives have been investigated for their activity against malaria, 225 A major issue in drug development is the related to safety and toxicity and the vast majority of novel compounds drop out from the developmental pipeline because of toxic effects to normal organs and tissues. Artemisinins are generally considered as safe in malaria treatment, although cases of toxicity also have been documented. 153,178,239 As the effective doses of artemisinin-type drugs against different diseases may differ, the safety profile of artemisinins in malaria treatment may not be identical for other diseases.
Considerably higher drug doses of artemisinins are necessary in cancer therapy. Preliminary clinical trials also indicate good tolerability, but again cases of toxic reactions became aware. 159,168,[240][241][242] As of yet, it is not known, which side effects would appear, if artemisinin-type drugs will be used for the treatment of anti-inflammatory or autoimmune diseases. Therefore, the conductance of preclinical toxicity studies and clinical Phase 1 trials are mandatory for the further development of artemisinin-type compounds to treat inflammatory and autoimmune diseases. However, the currently available data from mouse experiments did not report on major side effects. With all caution, it could thus be assumed that artemisinins may be also well tolerable to treat inflammatory or autoimmune diseases as described for malaria therapy.
Nevertheless, reports on toxic reactions should be taken Seriously. Relatively low concentrations are applied for a few days only for malaria therapy. Hence, drug accumulation over prolonged periods of time, which are frequently responsible for toxic side effects, is less probable. Furthermore, oral application is safer than intravenous or intramuscular injections. The best doses, treatment duration, and application routes have not been elaborated for EFFERTH AND OESCH | 3051 inflammatory and autoimmune diseases. Another rather unexpected aspect is that the combination of artemisinin with other drugs may not only lead to synergistic therapeutic effects toward the disease but may also cause antagonistic effects concerning toxicity, that is, to reduce side effects of other drugs. 179,180 All these aspects have to be carefully studied in clinical trials to come up with optimized treatment regimens against inflammatory and autoimmune diseases. In basic research, there are important tasks to be done. As exemplarily shown for some artemisinin-derived synthetic compounds, 166 it is within the scope of expectations to develop novel chemical entities with less toxicity. Hence medicinal chemistry should systematically investigate possibilities to generate novel artemisinin-related compounds with improved disease-fighting features at reduced toxicity.
Another word of caution is appropriate concerning the specific action of artemisinins toward immune cells.
Whereas the vast majority of investigations reported on the immunosuppressive effects of artemisinin-type drugs, there are also some studies that observed an increase of immune function, for example, by the inhibitory activity against Treg cells, the recovery of the Th1/Th2 balance, and so on. [243][244][245][246][247] This might be beneficial in cancer treatment, but counterproductive to treat inflammatory and autoimmune diseases. Here, immunosuppressive rather than immune-stimulating effects are required. It could be imagined that contrary effects might depend on divergent cellular or tissue-specific contexts in different diseases. This is a potential issue that has to be investigated and clarified in future experimentations.
To sum up in a nutshell, artemisinin-type drugs are a highly valuable class of drugs with high potentials for the treatment of inflammatory and autoimmune diseases. The available data in the literature justify to further investigations especially of randomized clinical trials to feed the drug development pipeline for the sake of patients suffering from inflammatory and autoimmune diseases.

ACKNOWLEDGEMENT
Open access funding enabled and organized by Projekt DEAL.